Prevalence of Parvovirus B19 and Parvovirus V9 DNA and Antibodies in Paired Bone Marrow and Serum Samples from Healthy Individuals

Parvovirus B19 (hereafter referred to as B19) exhibits a markedtropism to human bone marrow (BM), and infection may lead toerythema infectiosum, arthropathy, hydrops fetalis, and varioushematologic disorders. Recently, a distinct parvovirus isolatetermed V9 with an unknown clinical spectrum was discovered.In contrast to the many studies of B19 serology and viremia,valid information on the frequency of B19 or V9 DNA in the BMof healthy individuals is limited. To develop a reference value,paired BM and serum samples from healthy subjects were testedfor the presence of B19 and V9 DNA and specific antibodies.Immunoglobulin M (IgM) was not found in any of the serum samples.The prevalence of IgG showed a gradual and steady increase from37% in children aged 1 to 5 years to 87% in people aged >50years. When 190 well-characterized subjects were examined, B19DNA was detected in the BM of 4 individuals (2.1%; 95% confidenceinterval, 0.58 to 5.3%) while none of the paired serum samplesshowed evidence of circulating viral DNA. V9 DNA was not foundin any of the BM or serum samples. The finding of B19 DNA probablyindicated a primary infection in one 7-year-old individual andreinfection or reactivation of persistent infection in the remainingthree persons, aged 47 to 58 years. Serving as a benchmark forfuture studies, these findings are useful when interpretingepidemiologic data, performing BM transplantation, or consideringclinical implications of parvovirus infection.

INTRODUCTION

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Introduction

Human parvovirus B19 (hereafter referred to as B19) is a memberof the genus Erythrovirus of the family Parvoviridae (18). B19exhibits a marked tropism to human bone marrow (BM) and replicatesonly in erythroid progenitor cells (3). Infection may lead toerythema infectiosum, arthropathy, hydrops fetalis, and varioushematologic disorders, including aplastic crisis, chronic anemia,and idiopathic thrombocytopenic purpura (3). Diagnosis of B19relies on serology and the detection of viral DNA by PCR ordot blot analysis. A possible emerging parvovirus isolate termedV9 with an unknown clinical spectrum and markedly differentfrom B19 (>11% nucleotide disparity) was recently discovered(19). Sequencing, combined with PCR studies, has since demonstratedthe need for specific and differentiated techniques when examiningsamples for possible B19 or V9 viremia (11). Conversely, cloningand production of the V9 capsid proteins and subsequent enzyme-linkedimmunosorbent assay studies have revealed a 100% serologic cross-reactivitybetween the B19 and V9 isolates (13).

The prevalence of immunoglobulin G (IgG) antibodies directedagainst B19 ranges from 15 to 60% in children 6 to 19 yearsold: and from 30 to 60% in adults and is more than 85% in thegeriatric population (1, 7, 22). The frequency of B19 viremiain voluntary blood donors has been estimated at rates of 1:167to 1:35,000 (11, 14, 17, 22-24). As opposed to the many studieson B19 serology and viremia, information on the presence ofB19 DNA in the BM of healthy individuals is limited. Thoughno serum was available for comparison, B19 DNA was discoveredin 4 of 45 (9%) healthy bone donors (5), while another studyreported no evidence of B19 DNA in the BM of 13 BM donors (9).When paired serum and BM samples have been examined for B19DNA, it has been shown that serum samples are only positivein a fraction (0 to 50%) of patients with positive BM samples,which may be explained by the tropism of B19 (2, 5, 6, 15, 16).Likewise, monitoring the clearance of virus by detection ofDNA in sequential samples is also less sensitive when serumsamples are used rather than BM. Moreover, no study of the prevalenceof V9 in the BM of patients or in the general population exists.

To develop a reference value, we examined paired BM and serumsamples from healthy subjects for the presence of B19 and V9DNA and specific antibodies. This material may be used as apoint of reference for future studies of parvovirus infectionand when discussing possible persistent infection or reinfectionby this pathogen.

MATERIALS AND METHODS

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Materials and Methods

Materials. Paired serum and BM samples from 153 consecutive healthy DanishBM donors for transplantation, assembled from 1981 to 1989 (n= 24) and 1995 to 2000 (n = 129), were retrieved (Table 1). Themedian age was 28 years, with 38 samples available from individualsin the chief B19-susceptible age (1 to 20 years). The panelof samples was supplemented by BM samples (no sera were available)from 37 infants with sudden infant death syndrome (SIDS) obtainedfrom 1988 to 1995, which were believed to be unbiased in regardto parvovirus infection.

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TABLE 1. Age and gender characteristics of 190 individuals enrolled in the study

PCR procedure. All BM samples were initially tested by PCR for the presenceof part of the human ß-globin gene, ensuring the presenceof intact DNA material for subsequent parvovirus detection (10,20). Serum and BM samples were then examined by nested PCR,which involved consensus primers for the primary rounds of amplificationfollowed by separate primers for the amplification of B19 andV9 DNA, respectively (11). The nested assay had a consistentsensitivity and was able to correctly identify 1 to 10 copiesof target DNA in a 5-µl sample volume (200 to 2,000 copies/ml)(11).

Sequencing reactions. To rule out the possibility of contamination and to characterizethe viral strains, sequencing of a 390-bp fragment of the VP2gene (nucleotides 4071 to 4460) (21) in all positive samplesand the corresponding positive controls was performed as previouslydescribed (11).

Serologic tests. The presence of specific B19 and V9 IgM and IgG antibodies wasdetermined using V9 recombinant VP2 capsid proteins in a newlydeveloped enzyme-linked immunosorbent assay, which shows a 100%serologic cross-reactivity between the B19 and V9 isolates (13).

Morphology. Positive BM samples were reviewed histologically by one of theauthors (B.L.P.); morphologic features, including the presenceof megakaryocytes and giant pronormoblasts, were noted, andmyeloid/erythroid ratios were calculated.

Ethical considerations. The study was approved by The Scientific Ethics Committee ofCopenhagen, Denmark (reference 01-360/94).

RESULTS

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Results

PCR procedure. All positive controls were positive, and all negative controlsremained negative during the PCR experiments. The human ß-globingene was detected in every BM sample, allowing all of the initiallycollected samples to be included in the study. B19 DNA was detectedin four BM samples (2.1%; 95% confidence interval, 0.58 to 5.3%,or 2.6% in individuals >1 year old) (subjects 1 to 4), whilenone of the paired serum samples showed evidence of circulatingviral DNA (Table 2). V9 DNA was not found in any of the BM orserum samples.

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TABLE 2. Detection of parvovirus antibodies and specific DNA in blood and BM in 190 individuals enrolled in the study

Sequencing reactions. When sequenced and aligned to the reference B19 isolate pvbaua(21), each of the four B19-positive samples showed minor andindividual differences in the nucleotide sequence compared tothe B19 positive control (Table 3).

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TABLE 3. Sequence alignment of a 390-bp fragment of the B19 VP2 capsid gene (nucleotides 4071 to 4460) from four B19-positive clinical isolates and the positive control compared to the princeps isolate pvbaua

Serologic tests. B19 IgM was not found in any of the serum samples (Table 2).The prevalence of B19 IgG showed a gradual and steady increasefrom 37% in children aged 1 to 5 years to 87% in people aged>50 years, and the presence of B19 IgG was significantlycorrelated with higher age when compared to seronegative individuals(P = 0.009; Mann-Whitney rank sum test). All of the B19 DNA-positiveindividuals exhibited specific IgG.

Morphology. The cellularity of the specimens and the morphology of megakaryocyteswere normal. The myeloid/erythroid ratio ranged from 3:1 to5:1, and the erythropoietic cells were normal. Giant pronormoblastswere not noticed, and immunohistochemistry for parvovirus wasnegative in all cases.

Characterization of positive subjects. The ages of the B19-positive individuals were 7, 47, 56, and58 years, respectively, and none of them had received bloodproducts. Prior to BM transplantation, all donors were subjectedto a physical examination and a variety of serologic and morphologictests. All persons involved, including the four B19-positivedonors, were deemed healthy without signs of active or recentillness.

DISCUSSION

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Discussion

B19 is a ubiquitous infectious agent and is transmitted by therespiratory route or by blood and various blood products (3).Numerous studies of the occurrence of viral DNA and specificantibodies in the general population have been described (1,7, 11, 14, 17, 22-24). Simultaneous examination of BM and serumsamples from patients for the presence of B19 DNA has revealeddiscrepancies, with B19 being briefly or continuously detectedin BM following clearance of virus in the serum. The tropismand replication of B19 in BM and subsequent discharge of virusinto the circulation may explain the discordant results. Thus,detection of viral DNA in serum probably at best merely mirrorsa current infection taking place in the BM. Accordingly, inlow-grade or chronic infections, the viral load in serum maybe diminished to a degree below the threshold of detection,while BM samples remain positive. No major systematic data existon the prevalence of B19 or V9 in the BM of healthy subjects,let alone paired serum and BM samples. To generate a referencevalue, the prevalences of both B19 and V9 DNA and antibodieswere investigated using paired serum and BM samples from healthysubjects.

BM samples from 190 individuals were retrieved, and apart from37 children with SIDS, serum was available in all cases. Thenucleotide discrepancies detected by sequencing indicate thatthe B19-positive samples represent truly positive samples. B19has been known to undergo little genetic variation (1 to 2%of the genome) (8), and we found comparable genetic variationsin the B19-positive samples.

B19 DNA was not detected in any of the 153 serum samples (Table2), reflecting the fact that B19 viremia is an uncommon occurrenceamong blood donors. Likewise, the serologic results supportprevious findings in which the prevalence of IgG shows a gradualrise throughout life. Accordingly, in children aged 1 to 5 yearsapproximately one in three exhibited B19 IgG, while older childrenand adults had an even higher prevalence of 44 to 87% (Table2).

The prevalence of B19 and V9 DNA in the BM of the general populationhas hitherto been unresolved. Initial findings in two studiesinvolving only 45 (5) and 13 (9) people, with no serum samplesor clinical data available for comparison, showed discrepantresults, with the prevalence of B19 DNA in BM estimated at 0and 9%, respectively. Presently, when 190 well-characterizedsubjects were examined, B19 DNA was detected in the BM of 4individuals (2.1%), indicating that a baseline incidence ofB19 DNA of a few percent in the BM of the general populationis to be expected. Given the discrepant findings when pairedserum and BM samples were examined, as well as in the resultsfrom previous reports (2, 5, 6, 15, 16), it is likely, despitethe advent of the highly sensitive PCR technique, that a higherlevel of viral replication is necessary for detection of B19DNA in serum as opposed to BM.

The presence of viral DNA or specific IgM is generally acceptedas an indication of an ongoing or recent primary infection.Thus, detection of B19 DNA in subject 1 probably representedthe tail end of a primary infection, given the age (7 years)and the fact that no IgM was present. However, considering theages of the remaining three positive individuals (47 to 58 years),the finding of viral DNA may also represent reactivation ofpersistent infection or reinfection. Consequently, a mechanismanalogous to that of varicella might be possible, in which anage-related diminished level of antibodies or cell-mediatedimmunity can lead to low-grade reactivation or reinfection,with detectable levels of B19 DNA possibly found exclusivelyin the BM without accompanying overt clinical symptoms. It hasbeen proposed that B19 screening of blood components destinedfor children with malignancies be implemented (4), and thismay well include testing of BM donors prior to BM transplantation,given the present results and the discovery of symptomatic B19infection transmitted by BM (12).

In conclusion, B19 DNA may be detected in BM, but not serum,in a few percent of healthy individuals. Serving as a benchmarkfor future studies, these findings are of importance when interpretingepidemiologic data or considering the clinical implicationsof parvovirus infection.

ACKNOWLEDGMENTS

We are grateful to B. F. Vestergaard, Virology Department, StateSerum Institute, who collected stored clinical serum samplesfrom BM donors, and H. P. Hougen, Forensic Department, Rigshospitalet,for generously sharing BM material from SIDS children.

FOOTNOTES

* Corresponding author. Mailing address: IMMI, Panum Institute, Bldg. 24.2, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Phone: 45 35 32 76 79. Fax: 45 35 32 78 53. E-mail: e.heegaard{at}immi.ku.dk.

REFERENCES

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