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Journal of Virology, July 2007, p. 6927-6935, Vol. 81, No. 13
0022-538X/07/$08.00+0     doi:10.1128/JVI.02713-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biological and Immunological Relations among Human Parvovirus B19 Genotypes 1 to 3{triangledown}

Anna Ekman,1,{dagger},{ddagger} Kati Hokynar,1*,{ddagger} Laura Kakkola,1 Kalle Kantola,1 Lea Hedman,1 Heidi Bondén,1 Matthias Gessner,2 Claudia Aberham,2 Päivi Norja,1 Simo Miettinen,1 Klaus Hedman,1 and Maria Söderlund-Venermo1

Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital Laboratory, Helsinki, Finland,1 Plasma Analytics, Baxter AG, Vienna, Austria2

Received 8 December 2006/ Accepted 26 March 2007


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ABSTRACT
 
The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by ~10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1- or type 2-infected subjects (long-term immunity) was examined with homo- and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.


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INTRODUCTION
 
Human parvovirus B19, a member of the genus Erythrovirus within the subfamily Parvovirinae, has long been considered the only human pathogen of its family, in which the adeno-associated viruses of the genus Dependovirus conceivably are apathogenic. However, new parvoviruses distinct from the genus Erythrovirus were recently detected in plasma (PARV4 and PARV5) (20, 28) and in nasopharyngeal aspirates (human bocavirus) (1), the last of which is supposedly associated with severe respiratory illness in small children.

Although infection with parvovirus B19 typically results in erythema infectiosum or fifth disease (4), more severe or even lethal manifestations can occur among predisposed individuals. The virus replicates in erythroid progenitor cells of bone marrow (49, 64), causing aplastic crisis in patients with hemolytic anemia of various etiologies (2, 53, 56). During pregnancy, B19 can be transmitted from the infected mother to the fetus and cause fetal hydrops and death (9). In the immunocompromised, B19 infection may remain persistently productive, leading to chronic anemia (31).

The B19 virus is small and nonenveloped and encapsidates a linear single-stranded DNA genome of ~5.6 kb. The two genomic ends contain identical inverted terminal repeats of ~380 nucleotides that are imperfect palindromes and form hairpin loops (13). The genome contains only one functional promoter, p6, located in the 3' palindrome (15). The p6 promoter regulates the synthesis of nine RNA transcripts encoding the capsid proteins VP1 and VP2, the nonstructural protein NS1, and additional small proteins with incompletely known functions (36, 48, 65, 72).

The B19 DNA sequence was long considered extremely stable, with a variation of only ~1 to 2%. However, after recognition of the variant strains V9 (44, 45), A6 (46), and LaLi (27), the human erythroviruses are now classified into genotypes 1 (prototype), 2 (LaLi-like), and 3 (V9-like) (57). Furthermore, phylogenetic analyses have revealed two subgroups within genotypes 1 and 3 (52, 57, 67).

In overall sequence, these three types differ from each other by ~10%. The most striking variation is observed within the promoter area, in which the three virus types differ by >20%. Within the NS1 gene, sequence divergences between genotypes 2 and 3 and genotype 1 are ~13% at the nucleotide level and ~6% at the amino acid level. Within the open reading frame encoding the VP1/2 proteins, the majority of nucleotide substitutions are synonymous: at the nucleotide level, genotypes 2 and 3 differ from the prototype by ~9 and ~12%, respectively, but at the amino acid level they differ by only 1.1 and 1.4%. However, the degree of amino acid divergence within the VP1 unique region (uVP1) is higher: genotypes 2 and 3 differ from genotype 1 by 4.4 and 6.6%, respectively. Interestingly, amino acids 130 to 195 of the uVP1 gene containing the reported phospholipase 2 activity (16, 71) are highly conserved, and variation is mostly clustered in the N termini. Since important neutralizing epitopes are located within this region, differences in antibody response/recognition might ensue. Although a high degree of antigenic cross-reactivity has been shown between genotypes 1 and 3, almost no data has been available on the corresponding immunological relationship between genotypes 1 and 2 until the current study.

Postinfection, the DNA of the B19 prototype persists in solid tissues as an intact, continuous molecule devoid of any apparent persistence-specific mutations in the coding sequence (25). Furthermore, in our recent studies with over 500 samples of skin, tonsil, synovial, and liver tissues, the persistence of virus type 1 and 2 DNAs was shown to be frequent and lifelong (47), whereas persistent type 3 DNA was undetected in northern Europe. However, type 3 DNA has been encountered in blood endemically in Ghana and infrequently in France and Brazil (11, 44, 55, 57). Our recent studies (47) suggest that in northern Europe both virus types 1 and 2 circulated widely until the 1960s, after which type 2 disappeared and has subsequently occurred only sporadically. The genome substitution rate of B19 type 1, similar to that of canine parvovirus 2 (CPV2) (59), has been shown to be very high, approaching those of many RNA viruses (58). Whether the sudden disappearance of virus type 2 was due to stochastic variation, to immunological differences, or to retardation of biological proficiency or fitness in comparison to that of the type 1 virus is not known.

Because of the scarcity of data available on the biological activity of the B19 variant types 2 and 3 (5, 46), we set out to compare the biological and functional characteristics of the three virus types. This was carried out by examining the B-cell immunity, the p6 promoter activities, the NS1 trans-activating capabilities, the hemagglutination, and the infectivities of the three virus types.


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MATERIALS AND METHODS
 
Construction of p6 promoter-luciferase expression plasmids. The promoter region of B19 genotype 1, previously cloned into the luciferase-expressing plasmid pGL3enhancer (27), was excised and cloned into the KpnI and XhoI restriction enzyme sites in the plasmid pGL3basic (Promega, Madison, WI). The corresponding promoter regions of genotypes 2 (from a skin sample; LaLi) (27) and 3 (from viremic serum; D91.1) (57) were amplified by PCR (Table 1) and integrated into NheI and XhoI cloning sites of the plasmid pGL3basic. Inserts of the three promoter-luciferase constructs were sequenced at the core facility of the Haartman Institute (University of Helsinki, Helsinki, Finland) by the ABI PRISM technique.


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  		TABLE 1. PCR primers used in this study

Cell cultures. For measurement of the promoter strengths of the three virus types, the three p6 promoter constructs were transfected into several different cell lines. The human erythroid leukemia cell line KU812Ep6 (37), provided by E. Miyagawa (Fujirebio Inc., Japan); the human erythroblastoid cell line UT7/Epo-S1 (40, 60), provided by E. Morita (Tohoku University School of Medicine, Japan); and the human megakaryocytic leukemia cell line MB-02 (42), provided by D. Morgan (Hahnemann University), have been shown to be permissive for the B19 virus. In the monocytic cell line U937 (41), slow replication of erythrovirus type 1 was recently reported. For comparison, HuH-7 (human hepatocytes), HaCaT (human keratinocytes) (6), and HeLa (human cervical epithelial cells) represent B19-nonpermissive cell types. The HeLa derivatives HeLa/pGRE and HeLa/pGRE.NS (32), provided by F. Morinet (Hôpital Saint-Louis, Paris, France), were used for study of the trans-activating effects of the genotype 1 NS1 protein on the promoters of the three virus types. The latter cell line expresses the NS1 protein under the control of an inducible promoter (32).

HeLa and HuH-7 cells were maintained in modified Eagle's medium. HaCaT cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS). U-937 cells were maintained in RPMI 1640 containing 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 mg/ml glucose, and 1.5 mg/ml sodium carbonate. MB-02 cells were maintained in RPMI 1640 containing 10% human serum (B19 antibody negative) and 200 U/ml granulocyte-macrophage colony-stimulating factor (Sigma) and were differentiated by adding 4 U/ml erythropoietin (Eprex; Janssen-Cilag, Berchem, Belgium) and 25 ng/ml human stem cell factor (Sigma). The MB-02 cells were then grown for 5 days, and the differentiation was monitored by hemoglobin staining (alkaline phosphatase substrate kit IV; Vector Laboratories Inc., Burlingame, CA). KU812Ep6 cells were maintained in RPMI 1640 containing 10% FBS and 6 U/ml erythropoietin. UT7/Epo-S1 cells were maintained in Iscove's modified Dulbecco's medium containing 10% FBS and 2 U/ml erythropoietin. HeLa/pGRE and HeLa/pGRE.NS cells were cultured in modified Eagle's medium in the presence of 300 µg/ml hygromycin B (Sigma). The NS1 protein was induced by 1 µM dexamethasone (Sigma) (32) for 1 day prior to transfection. All media contained penicillin, streptomycin, and L-glutamine, and the cells were grown in 5% CO2 at 37°C.

Transfections and promoter activity assays. To measure the p6 promoter strength of each parvovirus type, their promoter regions were incorporated separately in vectors, using as a reporter firefly (Photinus pyralis) luciferase (Promega Corporation, Madison, WI). As an internal reference, production of a second type of luciferase derived from Renilla reniformis was achieved by cotransfecting the plasmid pRL-TK (Promega) to the cells, together with the promoter-luciferase constructs.

The suspension-cultured cells were transfected by electroporation during the exponential growth phase. For each reaction, 1 x 106 or 2 x 106 cells, 2 µg of a particular promoter-luciferase construct (Photinus), and 0.2 µg of the control plasmid pRL-TK (Renilla) were placed in a pulse chamber and pulsed (MB-02, 300 V and 960 µF; U-397, KU812Ep6, and UT7/Epo-S1, 350 V and 960 µF) with a Bio-Rad GenePulser (Bio-Rad Laboratories, Hercules, CA).

The adherent cells were seeded in the wells 1 day before transfection to reach 90 to 95% confluence. Transfection was done with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, except for HaCaT cells, in which the amount of Lipofectamine was 10 µl/12-well plate and 12.5 µl/35-mm2 plate. The amounts of reporter and internal reference used per transfection were 2.0 µg and 0.2 µg for HaCaT, 1.6 µg and 0.16 µg for HuH-7, and 0.8 µg and 0.08 µg for HeLa cells, respectively.

One day posttransfection, the cells were collected, washed with phosphate-buffered saline (PBS) (without Ca2+ and Mg2+), and lysed with Passive lysis buffer (Promega). A 20-µl sample of the lysate was used for quantification of the luciferase activity. Each sample was assayed in duplicate. The assays for the Photinus and Renilla luciferase activities were performed sequentially with a Dual-Luciferase Reporter assay system (Promega) as recommended by the manufacturer. The expressions of the test and the control reporters were measured with a Digene DCR-1 luminometer (MG Instruments Inc., Hamden, CT) in relative luminescence units (RLU) for a period of 10 seconds for each luciferase. The experimental RLU were normalized to the activity of the control RLU to minimize interreaction variability caused by differences in cell viability or transfection efficiency.

In all experiments, the vector pGL3control, which contains the firefly luciferase gene under the control of the simian virus 40 (SV40) promoter and enhancer sequences, was used as a positive control. To compare the activities of the different p6 promoter constructs within the different cell lines, the activity of the SV40 promoter was set at 1 in all cell types. The RLU of each virus type (normalized against the Renilla standard) was divided by the normalized RLU of the pGL3control. Water and the empty vector pGL3basic were used as negative controls. All transfection experiments were performed at least three times, and variation between independent tests is indicated by error bars (Fig. 1).


Figure 1
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  		FIG. 1. Comparison of the strengths of the p6 promoters of B19 types 1 to 3 in different cell lines. The values were calculated by dividing the amount of relative luciferase activity induced by each p6 promoter, normalized against the internal R. reniformis standard, by the relative luciferase activity induced by the SV40 promoter. The error bars indicate standard deviations.

B19 virus (types 1 to 3)-containing samples and hemagglutination assay (HA). The B19 genotype 1-containing single plasma donation SPR3 (DNA content, 4.5 x 1012 IU/ml) was obtained from the Finnish Red Cross Blood Transfusion Service, Finland (26). Genotype 2-containing serum IM-81 (DNA concentration, 5 x 1010 IU/ml), initially derived from a blood donor in Germany, was obtained from Baxter AG, Vienna, Austria (5). Genotype 3-containing serum D91.1 (DNA concentration, 1 x 1010 IU/ml), derived from a child with aplastic crisis in France, was obtained as a generous gift from A. Garbarg-Chenon, France (57). All samples tested negative for B19 antibodies.

In order to detect and compare the quantities of the viral particles in these samples, an HA was used (8). Recombinant capsids, produced with the baculovirus system, were used to set up the HA; human erythrocytes (obtained from the Finnish Red Cross Blood Transfusion Service) were washed three times and suspended (10% [vol/vol]) in PBS. The suspension was further diluted 1:20 in HA buffer (8 g/liter NaCl, 0.2 g/liter KCl, 5 g/liter dextrose, and 0.2% bovine serum albumin in 0.05 M PBS, pH 5.8), and 50 µl of the suspension was added to each well of a 96-well plate. Serial threefold dilutions of viremic sera were prepared in HA buffer, and 50 µl of each dilution was added to the plates and incubated for 2 hours at 4°C.

Infectivities of B19 virus types 1 to 3. KU812Ep6 (0.4 x 106cells/ml) (37) or UT7/EpoS1 (1.0 x 106 cells/ml) cells (40, 60) were suspended in medium containing viremic serum of type 1, 2, or 3. After incubation for 2 h at 37°C (KU812Ep6 cells) or 2.5 h on ice (UT7/Epo-S1 cells), fresh medium was added to yield a final density of 0.2 x 106 cells/ml. Infection was monitored on day 3 by immunofluorescence (IF) and reverse transcription (RT)-PCR. The viral capsid proteins were stained with a 1:1,000-diluted mouse monoclonal antibody (69), and the NS protein was stained with a 1:500-diluted human monoclonal antibody (22) for 60 min at room temperature in a moist chamber. The primary antibodies were then visualized by 1:30- or 1:50-diluted fluorescein isothiocyanate-conjugated secondary antibodies [polyclonal rabbit anti-mouse immunoglobulins (Dako A/S, Denmark) or AffiniPure F(ab')2 Fragment Goat anti-Human IgG (H+L) (Jackson Immunoresearch Laboratories, Inc., West Grove, PA), respectively] applied for 30 min. After being washed, the cells were mounted with Vectashield mounting medium containing DAPI (4',6'-diamidino-2-phenylindole) counterstain and observed with a Zeiss Axioplan 2 fluorescence microscope.

For RT-PCR, total cellular RNA was collected from the infected cells with Trizol reagent (Invitrogen) according to the manufacturer's instructions. The pelleted RNA was suspended in 30 µl of RNase-free water and stored at –70°C. RT-PCR was done with a RobusT RT-PCR II kit (Finnzymes, Espoo, Finland). The RT-PCR protocol, using B19L2 and B19S20 as primers for detection of all spliced B19 mRNA transcripts and Rb1 and Rb2O as control primers for detection of spliced mRNA of the cellular retinoblastoma gene, has been described previously (10).

Recombinant expression and purification of virus type 1 and 2 capsids. The production of virus-like particles (VLPs) of B19 type 1 in Spodoptera frugiperda Sf9 cells has been described previously (19, 29). Now, VLPs were synthesized from virus type 2, composed either of capsid protein VP2 alone or of proteins VP1 and VP2 together. The VP2 and VP1 genes of virus type 2 were amplified by PCR using as a template a virus type 2 clone (LaLi; nucleotides 199 to 5139, according to GenBank accession no. AY504945 of B19 isolate NAN) in a pSTBlue-1 vector (Novagen, Madison, WI). The primer sequences (primers KK1 to -4) are shown in Table 1.

The amplified VP1 and VP2 genes were cloned into the baculovirus transfer vector pAcuW51 (Pharmingen, San Diego, CA) under the control of the p10 and polyhedrin promoters, respectively. The recombinant VP2 and VP1/2 plasmids, together with linearized baculovirus DNA (Baculogold; Pharmingen), were cotransfected into Sf9 cells with Fugene6 transfection reagent (Roche Diagnostics, Indianapolis, IN), as described by the manufacturer. After 4 days, the supernatant containing the recombinant baculovirus was collected and plaque purified. The production of viral proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, and the formation of VLPs was verified by electron microscopy (EM). The recombinant VP2 or VP1/2 capsids of B19 types 1 and 2 were produced at large scale in High Five cells and were purified by 28% (wt/wt) CsCl gradient ultracentrifugation (100,000 x g for 48 h at +4°C), followed by precipitation with 40% ammonium sulfate.

EIA. For comparison of the antibody activities of B19 type 1- and type 2-infected subjects (defined by PCR of tissue biopsy specimens) (27, 47), four different enzyme immunoassays (EIAs), using as an antigen biotinylated VP2 or VP1/2 capsids of B19 type 1 or 2, were set up. The EIA procedure has been described previously (29). The optimal concentration of each antigen preparation (40 ng/well for type 1 VP2, 80 ng/well for type 1 VP1/2, 80 ng/well for type 2 VP2, and 100 ng/well for type 2 VP1/2) was first determined by end point titration. The four antigens were then tested with dilutions of the WHO international standard for anti-parvovirus B19 serum immunoglobulin G (IgG) (NIBSC 93/724). In addition, all antigens were tested with B19 acute-phase (low IgG avidity and acute epitope type specificity) and past-immunity (high IgG avidity and nonacute epitope type specificity) human serum control pools (29, 61, 62).

Serum samples for IgG cross-reactivity study. To examine the extent of IgG cross-reactivity between virus types 1 and 2, individual serum samples, derived from three groups of subjects, were studied. Group 1 contained sera of subjects persistently carrying B19 DNA of type 1 in skin and/or synovial tissue (n = 24), group 2 contained sera of subjects carrying B19 DNA of type 2 (n = 25), and group 3 contained sera of B19 IgG-negative subjects (n = 13). The group 3 IgG-negative samples were used to set the EIA cutoffs (the mean plus three times the standard deviation). All 62 serum samples were tested individually with all four antigens (altogether, 248 measurements). The range and mean value of absorbances gained with each of the four antigens were calculated for each patient group (Table 2).


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  		TABLE 2. IgG reactivities with VP2 or VP1/2 capsids of sera from subjects with B19 DNA of type 1 or 2 in tissue or from seronegative subjects

The subjects in groups 1 to 3 were healthy members of hospital or laboratory staff or patients with trauma or other non-B19-related disease and had been examined for B19 DNA persistence and genotyped by PCR methods described previously (27, 47). All the sera were studied for B19 IgG and IgM antibodies by in-house (29) and commercial (Biotrin, Dublin, Ireland) EIAs, respectively, and for VP2 IgG epitope type specificity (29, 62) and VP1 IgG avidity (61) for verification of long-term immunity.


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RESULTS
 
Promoter activity. To examine the impacts of sequence diversity within the p6 promoter region on the biological activity or cell tropism of the variant viruses, the p6 promoter strengths of all three virus types were measured in a variety of cell types in proportion to the strength of the SV40 promoter. The p6 promoter activities of the three B19 types were all of similar strength and exceeded that of the SV40 promoter in all cell types studied (Fig. 1). In MB-02 cells, the promoters of the three virus types were 35 to 41 times stronger than the SV40 promoter. In UT7/Epo-S1 cells, the promoters were 29 to 33 times stronger, and in KU812Ep6 and U937 cells, they were 11 to 17 and 14 to 24 times stronger than the SV40 promoter, respectively. In the nonpermissive HUH and HeLa cells, the promoter activities exceeded that of SV40 only four- to fivefold. In HaCaT cells, however, the promoter activity of virus type 1 exceeded that of SV40 27-fold, in comparison to an 8-fold increase of virus types 2 and 3. In other words, except in HaCaT cells, in which the type 1 promoter was ~3-fold stronger than those of genotypes 2 and 3, the promoters of the three B19 genotypes were of similar strengths (Fig. 1).

trans-activation of B19 promoters by the NS1 protein. The NS1 protein of B19 type 1 is known to enhance p6 promoter activity (21). To examine whether it could enhance the activity of p6 in all B19 virus types, the promoter-luciferase constructs were transfected into HeLa/pGRE.NS cells induced with dexamethasone to produce the genotype 1 NS1 protein (32). The expression of the NS1 protein was verified by IF. Uninduced HeLa/pGRE.NS cells and cells containing the native pGRE vector prior to (HeLa/pGRE) and following (HeLa/pGRE IND) induction served as negative controls. The promoter activities of the three virus types in the presence of NS1 were three to four times stronger than in the absence of NS1, with no significant difference between the three virus types (Fig. 2).


Figure 2
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  		FIG. 2. trans-activation of the promoter p6 constructs of B19 types 1 to 3 by the type 1 NS1 protein. The promoter p6 constructs of B19 types 1 to 3 were transfected with HeLa/pGRE.NS cells induced to produce the genotype 1 NS1 protein. For comparison, uninduced HeLa/pGRE.NS cells and cells containing the native pGRE vector prior to (HeLa/pGRE) and following (HeLa/pGRE IND) induction were transfected in parallel. The error bars indicate standard deviations.

Hemagglutination. The hemagglutination activities of viremic sera SPR3, IM-81, and D91.1 containing B19 virus types 1, 2, and 3, respectively, were measured. All of the virus types hemagglutinated the human erythrocytes at +4°C, pH 5.8, with HA titers of 14,400, 14,400, and 8,700. The HA titers corresponded to the respective DNA concentrations of 4.7 x 107 IU/ml, 1.7 x 106 IU/ml, and 5.5 x 105 IU/ml for the three viremic sera (Fig. 3).


Figure 3
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  		FIG. 3. HA of sera containing virus type 1 (SPR3), type 2 (IM-81), and type 3 (D91.1). The viral DNA concentration of each serum dilution is given as IU/ml.

Infectivities of virus types 1 to 3. To determine the viabilities of the three B19 genotypes, two myeloid cell lines, KU812Ep6 and UT7/Epo-S1 (Fig. 4), were infected with plasma/sera containing virus type 1, 2, or 3. All three genotypes were shown to be able to infect these cells. IF showed both NS1 and VP proteins in KU812Ep6 infected with virus types 1 to 3 and in UT7/Epo-S1 cells infected with virus types 1 and 2. In UT7/Epo-S1 cells infected with virus type 3, IF verified only capsid protein production due to scarcity of type 3 serum. RT-PCR showed the synthesis of spliced mRNA in all virus and cell types (data not shown).


Figure 4
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  		FIG. 4. VP1/2 staining (left) and DAPI counterstaining (right) in IF assays of KU812Ep6 and UT7/Epo-S1 cells infected with B19 virus types 1 to 3.

Expression of recombinant VP2 and VP1/2 VLPs of genotype 2. Sf9 cells were infected with recombinant baculoviruses containing the B19 virus type 2 gene(s) for either VP2 alone or VP1/2 together. With both constructs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent Western blotting verified the production of VP1 and VP2 proteins. EM of cell lysates showed icosahedral parvovirus-like particles with diameters of ~23 nm (Fig. 5). Both of these VLPs of virus type 2 hemagglutinated human red blood cells and were thus used, together with those of type 1, to optimize the HA.


Figure 5
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  		FIG. 5. EM of baculovirus-expressed purified type 2 capsids. Scale bar = 100 nm.

Immunological relationship of virus types 1 and 2. To examine the immunological relationship of B19 virus types 1 and 2, four IgG EIAs, using as an antigen VP2 or VP1/2 capsids of either virus type, were set up. Sera from three groups of subjects were examined with the four EIAs: group 1 contained sera from 24 subjects carrying B19 type 1 DNA in tissue, group 2 contained sera from 25 subjects carrying type 2 DNA in tissue, and group 3 contained sera from 13 B19 IgG-negative subjects. The results showed 100% cross-reactivity between B19 genotypes 1 and 2. The antigens of virus type 1 were recognized by the antibodies of virus types 1 and 2 with equal efficiency: the calculated means of absorbance values gained with the individual serum samples in groups 1 and 2 with VP2 antigen were 2.650 and 2.588, respectively, and with VP1/2 antigen they were 2.698 and 2.613, respectively (Table 2). The B-cell responses to the antigens of virus type 2 were similar: the means of absorbance values gained with serum samples in groups 1 and 2 with VP2 antigen were 1.038 and 1.049, respectively, and with VP1/2 antigen they were 2.189 and 2.142, respectively. With one sample (a type 2 serum), however, the type 2 VP2 EIA absorbance remained below threshold. The overall absorbances with this antigen remained lower than with the other three antigens, irrespective of the antigen concentration or of the genotype specificity of the serum used. All the antigens were equally reactive with the acute-infection and the past-immunity serum pools, as well as with the NIBSC International Standard for B19 IgG.


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DISCUSSION
 
B19 genotype 1 replicates restrictively in the erythrocyte precursors of bone marrow, yielding exceptionally high viral loads in blood during the early phase of infection (3, 18, 49, 64). In epidemiological studies with plasma pools from 100,000 Danish and 140,000 Finnish blood donors (23, 26) and in our recent studies of sera from 1,640 symptomatic patients, the DNA of B19 virus type 1, but not of type 2 or 3, was detected (47). By contrast, among blood donors in Ghana, the DNA of virus type 3 occurred frequently but in low copy numbers (11). Overall, high-titer viremias of virus types 2 and 3 have been encountered only sporadically (5, 33, 44, 46, 57). However, the DNA of B19 type 1 has been shown to persist in numerous tissue types, including synovial tissue, skin, liver, brain, muscle, and myocardial tissue, even though the mechanism of persistence and the cell type(s) involved are still unknown (reviewed in reference 63). Correspondingly persistent DNA of virus type 2 was encountered initially mainly in skin, pointing to a possible difference in tissue tropism between the B19 virus types (27). However, our recent studies of >500 tissues of various types revealed DNA of virus type 2, like that of the prototype, in all organs studied (47). Furthermore, for both of the B19 types, the persistence seemed to be lifelong. Moreover, in northern and central Europe, virus types 1 and 2 appear to have circulated equally until the 1960s, after which virus type 2 disappeared. By contrast, virus type 3 appears to have been absent from wide circulation in these areas during the past ≥70 years (47).

The molecular basis for these epidemiological differences is unknown, as are, in general, the biological functions and full pathogenetic potentials of virus types 2 and 3. Nguyen et al. (46) studied the infectivity of a virus type 2 isolate in UT7/Epo-S1 cells. No transcripts could be detected with RT-PCR. In contrast, Blümel et al. (5) observed equal mRNA expression of virus types 1 and 2 in KU812Ep6 cells. Until now, the biological activity of virus type 3 has not been studied. Prompted by the difference in the epidemiologies of the three B19 types, we examined the functional and immunological characteristics of the three virus types by a variety of interrelated approaches. The self-assembly of structural proteins into capsids has been shown for genotypes 1 (7, 30) and 3 (24). Here, we demonstrate the same feature for genotype 2 with the capsid proteins VP2 and VP1/2 expressed in the baculovirus system. Electron micrographs revealed icosahedral particles with the same diameter (~23 nm) as virus type 1 (12), which, furthermore, were able to hemagglutinate human erythrocytes. Under the same HA conditions, the type 1 to 3 viremic sera were shown, in addition to DNA, to contain virus particles with red cell surface-binding activity. The infectivity of these particles was demonstrated in two cell lines permissive for the parvovirus prototype. In both cell types, all three virus types induced transcription and maturation (splicing) of mRNAs and the synthesis of NS1 and VP proteins.

In our preliminary transfection experiments (27), the promoters of types 1 and 2 initiated transcription in UT7/Epo-S1 cells, but due to unequal construct lengths, the levels of gene expression could not be compared. To see if the high divergence within the p6 promoter affects the level of gene expression and the tropism of virus types 1 to 3, we examined the capacities of the respective full-length (21) promoters. The p6 promoters showed equal strengths, and the promoters were most active in permissive cell lines of myeloid origin. In nonpermissive cell cultures, originally derived from sites of recognized B19 DNA persistence (17, 27, 68), the p6 activities were lower but again equal between the three virus types, with the exception of an ~3-fold excess in the p6 activity of type 1 over those of types 2 and 3 in HaCaT cells, the meaning of which should be elucidated.

NS1 is a multifunctional protein and, at the amino acid level, is the most divergent protein among the three B19 genotypes. Its greatest variation resides in the amino-terminal portion, which contains a hypervariable region between amino acids 180 and 210 and the conserved motifs associated with single-strand nicking activities (14). The central region, containing the predicted ATPase/helicase (43) and nucleotide binding (38, 39) domains, is relatively conserved. NS1 acts as a transcription transactivator both by binding to the promoter DNA directly and via cellular transcription factors (54). We therefore examined the effect of the NS1 protein of virus type 1 on the promoters of B19 types 1 to 3 and found it to enhance equally the promoters of all three genotypes. It might still be possible that the divergent NS1 proteins of the three B19 types have some other genotype-specific characteristics preferentially functioning during replication or cell interaction that could possibly influence the viral fitness and the host cell range of B19.

A feature that has been associated with the host cell tropism of B19 is the relative production rates of the structural and nonstructural proteins in the infected cells (34, 70). This balance is determined by differential polyadenylation and splicing of the mRNA transcripts. Even though the internal polyadenylation sites, (pA)p1 and (pA)p2, of B19 pre-mRNAs are conserved between the three B19 virus genotypes, we have observed differences within the recently defined downstream and upstream cis-acting elements and the adjacent B motif influencing the activity of this polyadenylation site (70). Similarly, the sequence of the 11-kDa protein, which was recently shown to be critical for capsid protein production in the infected cells (72), differs between the three genotypes. These molecular divergences might contribute to the different epidemiologies (47), e.g., by influencing the tissue tropism and the persistence pattern. However, in that case this influence would need to be subtle, because we found both variant genotypes to produce capsid proteins and to infect the B19 type 1-permissive cells. Furthermore, both variants have been associated with anemia or aplastic crisis, indicating erythroid tropism (45, 46, 57).

On the other hand, the specific absence of virus type 2 from the tissues of the young (47) could be argued to be due to an immunological difference between the B19 types. In that scenario, a hypothetical mechanism for the maintenance of the bioportfolio would be equilibrium between viral replication and the efficiency of host immunity. However, phylogenetic analyses have shown the B19 nucleotide substitutions to be mostly synonymous and the substitution rates of the VP1 and NS1 genes to be similar, suggesting that immune selection is not the primary driving force of B19 virus evolution (58). Virus types 1 to 3 differ, at the protein level, by only 1 to 2% within the capsid, although most variation resides within the unique portion of the minor capsid protein VP1, which contains important neutralizing epitopes. Studies of antigenic relationships between B19 types 1 and 3 (24, 51) have shown a high degree of cross-reactivity, and in vitro neutralization tests using sera from subjects infected with B19 type 1 have shown inhibition of in vitro infection of type 2 (5). However, due to a general lack of methods to recognize type 2 IgG-positive sera and of type 2 recombinant antigens, until now it has not been possible to test whether sera from subjects infected with type 2 would react with antigens of type 1 or whether type 2 antigens are recognized equally well by type 1 sera. Because of the unique material of this study, consisting of sera from subjects infected with either type 1 or type 2 B19 virus (as evidenced by persistence of the respective B19 DNAs in tissue), and of our recombinant VLPs of both virus types and of both capsid proteins, we were able to address this question. In our study, 100% cross-reactivity in both VP2 and VP1/2 antibody EIAs between B19 types 1 and 2 was seen. Such knowledge of cross-reactivity is relevant for both diagnosis and vaccine development, as well as for definition of the taxonomic status of these human erythrovirus variants (66).

Taken together, the B19 virus types 1 to 3 were thus identical in all the experiments described above. Our results show that, even though virus types 2 and 3 are rare and in most studies only their DNA has been found, they indeed are biologically active viruses with the capacity for mRNA production and expression of both nonstructural and structural proteins, leading to formation of infectious virions. In agreement with recent studies showing similar kinetics for mRNA expression of types 1 and 2 in cell culture (5), our results show similar transcription efficiencies for the p6 promoters of all three virus types. Furthermore, the comparison of p6 activities in various cell types suggests similar tropisms. However, the striking differences in their temporal occurrences and geographical distributions (47) still await explanation. The cessation of B19 type 2 from circulation might be a fitness-related phenomenon resembling the replacement of CPV2 by the more effective lineage CPV2a (50, 59). Additional investigations, including measurement of the phospholipase A2 activity (16, 35, 71), definition of the additional functions of NS1, and elucidation of the impact of the 7.5- and 11-kDa proteins (72) and the polyadenylation regulation sites (70), may be needed to identify the molecular background of the epidemiological differences between the three B19 virus types. However, by a wide panel of functional, structural, and immunological approaches, our study has shown that the three B19 genotypes are, despite their sequence and epidemiological differences, highly similar variants of the same species. Furthermore, our results demonstrate that they constitute a single serotype.


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ACKNOWLEDGMENTS
 
We thank Eiji Morita, Kazuo Sugamura, Eiji Miyagawa, Doris Morgan, and Frederic Morinet for cell lines; Antoine Garbarg-Chenon for providing us with the B19 type 3 viremic serum sample; Susanne Modrow for the anti-NS1 human monoclonal antibodies; and Kazuo Sugamura for anti-VP mouse monoclonal antibodies.

This study was supported by the Paulo Foundation, the Jenny and Antti Wihuri Foundation, the Maud Kuistila Memorial Foundation, the Alfred Kordelin Foundation, the Ella and Georg Ehrnrooth Foundation, the Medical Society of Finland (Finska Läkaresällskapet), the Finnish Academy (project 76132), the Commission of the European Community (QLK2-CT-2001-00877), the Helsinki Biomedical Graduate School, and the Helsinki University Central Hospital Research and Education Fund.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), University of Helsinki, Helsinki, Finland. Phone: 358-9-19126676. Fax: 358-9-19126491. E-mail: Kati.Hokynar{at}helsinki.fi Back

{triangledown} Published ahead of print on 4 April 2007. Back

{dagger} Present address: Division of Anatomy, Department of Basic Veterinary Sciences, University of Helsinki, Helsinki, Finland. Back

{ddagger} A. Ekman and K. Hokynar contributed equally to this study. Back


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Journal of Virology, July 2007, p. 6927-6935, Vol. 81, No. 13
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