| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute of Virology, University of Bonn, Sigmund-Freud-Straße 25, D-53105 Bonn, Germany
2 Department of Surgery, University of Bonn, Sigmund-Freud-Straße 25, D-53105 Bonn, Germany
3 Institute of Pathology, University of Bonn, Sigmund-Freud-Straße 25, D-53105 Bonn, Germany
4 Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, D-63225 Langen, Germany
Correspondence
Anna Maria Eis-Hübinger
Anna-Maria.Eis-Huebinger{at}ukb.uni-bonn.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the sequences 30.1, 58, 59, 60 and 30.3, and 31.2, 32.2 and 33.2 are DQ408301–DQ408305 and DQ333426–DQ333428, respectively. The accession numbers for the genotype 1 sequences 1–10 and 12–29 as well as for the genotype 2 sequences 34–57 read as follows: EU144308–EU144317, EU144318–EU144335 and EU144336–EU144359, respectively.
Supplementary tables are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In adults, infection often manifests as symmetrical arthropathy, which may persist (Woolf et al., 1989). Due to viral replication in the erythroid progenitor cells, severe or life-threatening diseases can occur in immunocompromised patients and in individuals with shortened red cell survival (Brown et al., 1984; Kurtzman et al., 1988; Pattison et al., 1981; Public Health Laboratory Service, 1990; Skjöldebrand-Sparre et al., 2000). Besides the typical features, B19V is implicated in a wide spectrum of illnesses including neurological, cardiac, hepatic and rheumatological disorders (Broliden et al., 2006; Corcoran & Doyle, 2004; Heegaard & Brown, 2002; Kerr et al., 2002; Lehmann et al., 2003; Young & Brown, 2004).
The B19V genome consists of a single-stranded DNA, approximately 5.6 kb in length that encodes three large proteins (Clewley, 1984; Cotmore et al., 1986; Deiss et al., 1990). The non-structural protein NS1 is composed of 671 aa, the capsid proteins VP1 and VP2 are composed of 781 and 554 aa, respectively. Since the reading frames of VP1 and VP2 are in-frame, the two proteins are identical except for the additional unique portion of VP1 (VP1u) at its amino-terminal end (Ozawa et al., 1987).
Recently, it has been shown that the genetic diversity of B19V is higher than previously expected, resulting in subdivision of the species into three distinct genotypes (Gallinella et al., 2003; Hokynar et al., 2002; Nguyen et al., 2002; Servant et al., 2002). All viruses previously termed as B19V were classified as genotype 1. The nucleotide divergency between the genotypes is approximately 10 % and in the promoter region more than 20 %. Additionally, genotype 3 viruses cluster into two subtypes represented by the prototype strains V9 (GenBank accession no. AX003421
[GenBank]
) and D91.1 (GenBank accession no. AY083234
[GenBank]
) (Parsyan et al., 2007).
Until recently, genotype 2 viraemic individuals were found relatively infrequently (Blümel et al., 2005; Cohen et al., 2006; Liefeldt et al., 2005; Nguyen et al., 1998, 1999, 2002; Servant et al., 2002). Even in plasma factor concentrates, produced from thousands of blood donations, genotype 2 DNA was only detected in a very small percentage of individuals (Schneider et al., 2004). In contrast, genotype 2 DNA was found at a much higher frequency in tissue from individuals older than approximately 40 years of age, leading to the assumption that genotype 2 has widely disappeared from circulation (Norja et al., 2006). Genotype 3 virus was shown to be endemic in Ghana, West Africa (Candotti et al., 2004) and may be present in a certain region of Brazil (Sanabani et al., 2006). Outside these areas, only a few sporadic cases of viraemic infection have been reported in France (Nguyen et al., 1998, 1999; Servant et al., 2002) and one case was identified in the UK (Cohen et al., 2006). In an investigation performed in the USA, even the persisting genotype 3 DNA has only been found in a small percentage of tissue samples (Wong et al., 2003).
One of the main questions raised by the discovery of the new viral variants is whether the similarity between the genotypes results in restriction of infection to one genotype or whether multiple infections are possible. To answer this question, we examined liver tissue samples from adults for the presence of B19V DNA of genotypes 1, 2 and 3. The liver tissue was chosen because it harbours persisting DNA at a high frequency (Eis-Hübinger et al., 2001; Norja et al., 2006). The results presented here show that a small proportion of B19V-infected livers contains DNA of two genotypes, indicating double infection. In double-infected specimens of genotypes 1 and 2, a repertoire of genotype 1 genome variants was detected.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
B19V serological assays.
Anti-B19V IgG and IgM antibodies were measured in sera using an ELISA based on baculovirus-expressed VP2 (Biotrin).
PCR for screening.
DNA was prepared by spin-column procedure as described previously (Eis-Hübinger et al., 2001). Nested PCR for the detection of genotype 1 DNA was performed as described earlier (Eis-Hübinger et al., 2001). Nested PCR for the detection of genotype 2 DNA was carried out as described elsewhere (Schneider et al., 2004). Primers for the amplification of genotype 3 DNA were as follows (5'–3', positions according to GenBank accession no. AX003421
[GenBank]
): outer forward, nt 2235–2254; outer reverse, nt 2511–2488; inner forward, nt 2260–2279; inner reverse, nt 2374–2355. PCR conditions were identical to genotype 2 PCR except for annealing at 55 °C. Positive and negative controls were included in every run. Strict precautions to avoid contamination were taken. The sensitivity of the PCRs was less than two genome equivalents per reaction. Since especially the specificity of the genotype 1 PCR against genotype 3 virus was limited, genotyping of all viruses was performed by extended DNA sequence analysis. The results of the screening PCR for genotypes 1 and 2 from 81 and 77 liver samples, respectively, and for genotype 1 from the six bone marrow specimens have been reported (Eis-Hübinger et al., 2001; Norja et al., 2006).
DNA sequence analysis.
Nucleotide sequence analysis of the viral genomes was performed by direct sequencing of amplicons generated by nested PCR. Amplification of genotype 1 genome fragments was done with the primer sets NS1-C, 
V and VP1/VP2 (Hemauer et al., 1996), V-IIIG1 and VPintG1 (Schneider et al., 2004), and the primer sets given in Supplementary Table S1 (available in JGV Online). Amplification of genotype 2 DNA was performed with the primer sets NS1-CG2, VG2, VP1/VP2G2, VPintG2, VPCG2 and VPextG2 (Schneider et al., 2004), and the primer sets shown in Supplementary Table S3 (available in JGV Online). Primer sets for genotype 3 are given in Supplementary Table S4 (available in JGV Online). Unless otherwise indicated, PCR was performed by using the Expand High Fidelity PCR system (Roche; enzyme A). Additional enzymes used were Easy-A High-Fidelity PCR Cloning Enzyme (Stratagene; enzyme B), BD Advantage-HF 2 kit (BD Biosciences Clontech; enzyme C) and PfuUltra High-Fidelity DNA Polymerase (Stratagene; enzyme D).
Nested PCR products were purified by QIAquick PCR Purification kit (Qiagen) and sequencing reactions were carried out using approximately 5–20 ng of the purified product. If not otherwise indicated, amplicons from at least two independent PCR reactions were sequenced in the forward and reverse directions using the nested primers. Sequencing was performed with the ABI Prism BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). Reactions were run on an ABI Prism 3130 (Applied Biosystems). Sequencing data were reviewed manually. Alignments were generated by CLUSTAL_X version 1.81 and sequence editing was performed using BioEdit (v7.0.5). In the case of double-infected samples, the sequence of each amplicon was individually aligned. DNA distance matrices were generated by using BioEdit. Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987). To confirm the reliability of the phylogenetic trees, bootstrap resampling tests were performed 1000 times (Felsenstein, 1981). Visualization of the phylogenetic tree was performed using TreeView (version 1.4; http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
For detailed analysis of genome 31.1, the nucleic acid preparation was serially twofold diluted in sterile pharmacy water down to 1 : 1024 and dilutions subjected in triplicate to PCR using the primer set V-IIIG1. Additionally, the nucleic acid preparation was diluted 1 : 5 and subjected to PCR in quadruplicate.
Cloning of PCR products and mixing experiments.
Genotype 1 PCR products were generated from plasmid pGEM-1/B19 using the outer primers V-IIIG1. The plasmid was kindly provided by Dr Jonathan P. Clewley, Central Public Health Laboratory, London, UK. Genotype 2 PCR products were generated from genotype 2 virus IM-81 (Blümel et al., 2005) using the primer pair V-IIIG2 [forward, nt 2123–2142 (5'–3', positions according to GenBank accession no. AY044266
[GenBank]
); reverse, nt 3332–3313]. The amplified regions were homologous to each other. The PCR products were cloned into the pCR4-TOPO plasmid (Invitrogen), resulting in the plasmids pV-IIIG1 and pV-IIIG2. The inserts were sequenced with M13 forward and reverse primers.
For the mixing experiments, purified plasmid preparations were adjusted to the same concentration. Serial 10-fold dilutions up to 10–10 (approx. 3.2x109–3.2 copies µl–1) of plasmid pV-VIIIG1 were prepared in a nucleic acid eluate obtained from a B19V DNA-negative liver specimen. Each plasmid (1 µl) p-VIIIG1 dilution was mixed with 1 µl of plasmid pV-IIIG2 solution held at constant concentration (approx. 3.2x109 copies µl–1) and subjected to nested PCR using primer set V-IIIG1. Except for a template volume of 2 µl in the first round, PCR was performed as described previously (Schneider et al., 2004). After purification, the inner product was sequenced. The PCR reliably detected 2.5 genotype 1 genome equivalents per reaction. Genotype 2 DNA was amplified when present as the sole genotype in the reaction in a concentration of approximately 
4x104 genome equivalents per reaction as determined by plasmid titration.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The sequenced region of genotypes 1 and 2 DNA spanned from nt 1895 to 3064 (numbering according to genotype 1 prototype strain Au); sequencing was performed by using the primer sets NS1-CG1 and VG1/V-IIIG1 (the latter one for two specimens for which no amplicons could be generated by V) and the primer sets NS1-CG2 and VG2, respectively. Genotype 3 genomes were sequenced from nt 36 to 4967 (numbering according to genotype 3 subtype D91.1) with the primer sets given in Supplementary Table S4 (available in JGV Online).
|
). Four of the infected specimens showed double infection. In three specimens, double infection with genotypes 1 and 2 was detected, in one specimen double infection with genotypes 1 and 3 was detected. At least one genotype was present in 59 of 87 (67.8 %) specimens.
|
Extended sequence analysis also reproducibly revealed heterogeneities at single nucleotide positions, i.e. ambiguities (simultaneous detection of different nucleotides as double peaks in the chromatogram) as well as separate detection of each nucleotide observed at that position. The heterogeneities were observed at the same positions in different sequence reactions performed on independently generated PCR amplicons. The total number of polymorphic positions in genomes 31.1, 32.1 and 33.1 was 49, 63 and 64, respectively (1.00–1.31 % of the nucleotides). The polymorphisms were scattered throughout the whole of the sequences. Except for one position in genome 32.1 and one position in genome 33.1, the polymorphisms were represented by two bases (i.e. A/G, C/T, C/A, T/G, A/T or G/C). At position (Au) 924 of genome 33.1 and position 1392 of genome 32.1 three bases (A/T/C) were found to represent the polymorphisms (Table 1).
|
By comparison with prototype strain Au and the 25 longest genotype 1 genome sequences available in the database (strains Vn115 and Vn147 not considered), we analysed whether the heterogeneities were at positions where genotype 1 displays a high degree of divergence (Table 1
). Alignment showed that at 67 of 105 (63.8 %) polymorphic positions all comparison strains had an identical nucleotide. Interestingly, at 23 of 67 (34.3 %) positions two or all three polymorphic isolates displayed the same sequence polymorphism.
The most frequent polymorphism observed was A/G (43 positions), the second frequent polymorphism was C/T (38 positions). A/G was present at positions where the comparison strains displayed A or G except for two positions where the majority of comparison strains displayed G but a minority showed T. C/T was present at positions represented by T or C in the comparison strains except for one position where one comparison strain displayed G.
Only a small proportion of the polymorphisms in genomes 31.1 and 33.1 resulted in changes of the deduced amino acids of the large viral proteins. The ratio of non-synonymous to synonymous changes was 0.0625 (genome 31.1) and 0.1667 (genome 33.1) for the non-structural protein NS1, and 0.0870 and 0.0313 for the minor capsid protein VP1, respectively. Although the proportion of polymorphisms in the VP2-coding sequence was not significantly lower than in NS1- and VP1u-coding sequences, no changes would occur in VP2. In genome 32.1, the proportion of non-synonymous substitutions resulting from nucleotide polymorphism was higher and changes in the deduced amino acid sequences were present in each protein (ratio 0.3500, 0.3000 and 0.1875 for NS1, VP1 and VP2, respectively).
By direct sequencing the genotype 1 sequence derived from the genotypes 1 and 3 co-infected specimen (sequence 30.1) no polymorphism was observed. At least three independent PCR and sequencing reactions of each target region were performed using the primers sets given in Supplementary Table S2 (sequenced region, nt 158–5047). Nucleotide heterogeneity was also not observed in genotypes 2 and 3 sequences from the double-infected specimens [sequenced region of the genotype 2 genomes, nt (Au) 146–5077 (genome 31.2), 309–5077 (genome 32.2), and 309–4830 (genome 33.2)]. Direct sequencing of the genotype 2 genomes was performed with the primer sets NSNIG2, NSNIIG2, VP1/VP2G2, VPintG2, VPCG2 and VPextG2 (all genomes), FinG2 (genomes 31.2 and 32.2), and ProG2 (genome 31.2), respectively.
Sequence analysis showed that genotype 2 genome 32.2 displayed greater divergence to the respective prototype strains than the other genotype 2 genomes (distance matrices to strains LaLi and A6, 0.0396 and 0.0443, respectively). Alignment of the genotype 3 sequences with the respective prototype strains showed that all four genomes were more closely related to subtype D91.1 than to subtype V9. Interestingly, at the deduced amino acid level of the structural proteins VP1 and VP2, the DNA isolates were, however, more closely related to V9. Among the large B19V proteins NS1, VP1 and VP2, the genomic divergence to V9 and D91.1 was highest in the sequence encoding the main viral capsid protein VP2; however, was lowest at the deduced amino acid level.
Genetic diversity among the genotypes within a patient
Nucleotide sequence divergence (distance matrix) between the genotypes within an individual were 0.1453 (genotype 1 vs genotype 3), and 0.1323, 0.1249 and 0.1172 (genotype 1 vs genotype 2). Thus, the divergency rates were in the typical range of discrepancy between B19V genotypes. For the genotype 1 sequences, minimum genetic distance was calculated because of the ambiguities.
In the region between nt (Au) 309 and 4830, the sequences of all three pairs of genotype 1 and 2 viruses were available. The number of nucleotides differing between the genotypes within a pair was 544, 529 and 537, respectively. Genotype 1 heterogeneities were observed at 47, 55 and 60 positions, respectively. The majority of heterogeneities (60, 55 and 57 %) were located at positions where the reference strains Au and LaLi as well as the genotype 2 genome originating from the same specimen exhibited the same nucleotide. The genotype 1 and 2 reference strains differed by 511 positions. Thus, it seemed unlikely that the polymorphisms observed in the genotype 1 sequences were due to co-amplification of genotype 2 DNA. This assumption is supported by the fact that in all three genotype 1 genomes an ambiguity was observed in the promoter region between nt (Au) 310 and 314. In all three genotype 2 genomes the corresponding positions were deleted.
Analysis of sequence variation
To verify the phenomenon of sequence polymorphism, genome 31.1 was amplified by primer set V-IIIG1 using three additional enzymes (B, C and D) followed by direct nucleotide sequence analysis. With enzymes B and C four independent PCR and sequencing reactions, for each enzyme, were performed (B1–B4 and C1–C4). With enzyme D an amplicon was obtained only in one reaction (D1). With the enzyme used so far (A), eight independent reactions (A1–A8) were carried out. Clearly readable sequences were obtained in all reactions between nt (Au) 2247 and 3284.
Using enzyme A, nucleotide polymorphisms were observed at 13 positions (Fig. 3). Using the enzymes B and C, polymorphism was observed at the same positions, though not as ambiguities, i.e. as double peaks in the chromatogram. Thus, the results indicated that the polymorphism observed in the genotype 1 genomes was unlikely to be due to inaccuracy of enzyme A used so far. Four types of unambiguous genotype 1 sequences could be differentiated: type I, sequences A4–A6, B2–B4, C2–C4 and D1; type II, sequences A1 and A2; type III, sequence B1; type IV, sequence C1. Except for the 13 polymorphic positions, all other positions differing between genotype 1 and 2 from specimen 31 showed identical nucleotides, irrespective of the enzyme used. Fig. 3 shows the results for all positions (n=72) where the reference strains Au and LaLi and the genomes 31.1 and 31.2 were not homologous to each other.
|
V-IIIG1. Amplicons were yielded in one out of three reactions performed on the dilutions 1 : 2, 1 : 4 and 1 : 32, respectively, and one out of four reactions performed on the dilution 1 : 5. Nucleotide analysis of the amplicons revealed three of the four known sequence types without ambiguities (Table 2). This observation supports the hypothesis that three or more variants of genotype 1 genomes are present in the same tissue.
|
V-IIIG1 and primers V-IIIG2, respectively. Equal volumes of serially 10-fold diluted plasmid pV-IIIG1 (approx. 3.2x109–3.2 copies) and constant amounts of plasmid pV-IIIG2 (approx. 3.2x109 copies) were mixed and subjected to first and second round PCR using the primer set V-IIIG1 followed by sequencing.
All sequences were clearly readable between nt (Au) 2248 and 3278. Within this region, the genotypes 1 and 2 differed by 52 positions. Sequencing showed that the sequences obtained up to the dilution of 10–4 of pV-IIIG1 (approx. 3.2x106 copies) were unambiguously genotype 1. At pV-IIIG1 dilutions 10–5–10–10 (approx. 3.2x105–3.2 copies) double peaks were seen in the chromatogram at all positions the two genotypes differed from each other. Thus, the experiments showed that the pattern of nucleotide polymorphism due to co-amplification of genotype 2 is different from that observed in the polymorphic genotype 1 genomes, revealing the majority of polymorphisms at positions identical between the genotypes. Furthermore, the results showed that the polymorphic genotype 1 sequences presented fewer ambiguities than would be expected from co-amplification of two genotypes.
In summary, the results showed that three or more genotype 1 genome variants were present in the same tissue.
Relationship between B19V DNA detection and antibody status
Adequate sera were available from 71 individuals. Fifty-nine sera were B19V IgG-positive. B19V DNA, irrespective of its genotype, was present in 45 (76.3 %) liver specimens from seropositives. The detailed detection rates for genotypes 1, 2 and 3 DNA were 39.0, 37.3 and 3.4 %, respectively. Except for one specimen containing genotype 2 DNA, no B19V DNA was found in the samples from the 12 seronegatives. All sera tested negative for B19V IgM.
Identical B19V genome sequences at various body sites
Bone marrow specimens from six individuals were tested by PCR for genotypes 1, 2 and 3 DNA followed by sequence analysis (Table 3). In two individuals already found to host genotype 2 DNA in the livers, genotype 2 DNA was also detected in the bone marrow. Viral sequences within one individual were identical. One of these individuals was B19V-seronegative as described above. Identity of the B19V DNA at different body sites was further observed in an individual with genotype 1 persistence. Sequencing was done from nt (Au) 1912 to 3023 using the primer sets NS1-C and V or NS1-CG2 and VG2, respectively.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Eis-Hübinger et al., 2001; Söderlund et al., 1997; Wong et al., 2003). After acute infection, residual viral DNA can remain in tissue for decades or even lifelong (‘bioportfolio’) (Norja et al., 2006). Herein, we report on the co-persistence of different genotypes and several genomic variants belonging to the same genotype.
Of the 59 specimens hosting B19V DNA of either genotype, simultaneous persistence of two genotypes was detected in four (7 %) samples. Although the number of cases with double B19V infection is limited, the question is pertinent as to how infection with two genotypes can occur. At present, we can only speculate about possible explanations. The favoured hypothesis is that infections were acquired sequentially (superinfection). However, this would implicate that, at least in a certain percentage of individuals, infection with one genotype does not confer sufficient protective immunity against infection with another genotype. Since especially neutralizing antibodies are essential for clearance of acute infection and confer lasting protection (Anderson et al., 1985; Corcoran et al., 2004; Kurtzman et al., 1989), a weaker than usual neutralizing antibody response might be a reason why superinfection can occur. In a study by Blümel et al., 2005, genotype 2 virus was cross-neutralized in vitro by sera from patients having recovered from acute genotype 1 infection. However, in sera from some patients, neutralizing activity was somewhat lower for the heterologous genotype. Concerning the route of infection, transmission of B19V via respiratory secretions is the most common way. Concerning the fact that double infection was detected in four of 59 specimens positive for B19V DNA, this way of transmission seems most obvious to us. Alternatively, transmission of the second virus might have occurred via blood transfusion circumventing the natural barrier for entry. Recently, a case of short, low-level viraemia after exposure to a contaminated blood cell preparation in an already seropositive leukaemia patient was described, indicating that superinfection is possible in principle although rare (Plentz et al., 2005). Though not very likely, the possibility of concomitant infection with two genotypes cannot be completely ruled out.
Sequence analysis revealed nucleotide polymorphism in three genotype 1 genomes. Cloning of amplicons used for direct sequence analysis into the pCR4-TOPO plasmid followed by sequence analysis of individual clones confirmed the sequence variants. Cloning of the PCR product A5, which displayed an unambiguous sequence, by direct sequencing (Fig. 3), resulted in all clones displaying the same nucleotides as were observed by direct sequencing at the positions with polymorphisms. Furthermore, the sequence obtained by direct sequencing was identified in a clone. Similarly, cloning of amplicon B3, which also displayed by direct sequence analysis an unambiguous sequence that was identical to the sequence A5 (sequence type I), resulted in all clones showing the same nucleotides as those found by direct sequencing. In contrast, cloning of PCR product A7, displaying ambiguities by the direct sequencing approach, resulted in clones which showed either one or the other version of bases at the respective positions. One of four clones sequenced revealed sequence type IV, thus was identical to sequence C1 obtained by direct sequencing using another enzyme (enzyme C). Cloning of PCR reaction A3, which also displayed ambiguities, resulted in a clone that displayed one alternative of the ambiguities. This cloned sequence was identical to the sequences A1 and A2 obtained by direct sequencing (sequence type II).
Comparison with genotype 1 DNA sequences available in the database showed that the heterogeneities were not at positions displaying a high degree of nucleotide divergence. Surprisingly, a substantial proportion of polymorphisms was present at identical positions in two or three genomes and, additionally, displayed the same type of heterogeneity. This phenomenon and the fact that the pattern of nucleotide segregation was reproducibly observed at the same positions suggest that the polymorphism is more likely to represent a heterogeneous population of genotype 1 DNA molecules belonging to the same viral genotype than artefacts. However, the finding that polymorphism was not seen in specimens other than those doubly infected by genotypes 1 and 2 does not imply the strict absence of polymorphism in singly infected specimens since a critical amount of the variant sequence is necessary for being detectable by direct sequencing. Furthermore, we are aware that possibly not all variants may be recognized by the direct sequence analysis.
The mechanism by which persistence of several variants of genomes belonging to the same genotype is accomplished is currently unknown. Analogous to simultaneous persistence of two genotypes, reinfection might occur. However, since there was evidence for co-persistence of three or more variants, repeated reinfection has to be assumed. Another explanation could be that the genetic diversity has evolved during the acute infection. Especially during the initial phase of B19V infection, characterized by high levels of viral replication, there might be a greater chance of appearance of inaccurately replicated B19V DNA; although, the viral DNA is replicated by the host cell machinery. It has been recently speculated by others (López-Bueno et al., 2003; Shackelton & Holmes, 2006) that the fidelity and the proofreading activity of the enzymic complex containing the host DNA polymerase, recruited cellular replication factors and the viral NS1 protein may not be as efficient in single-stranded DNA viruses as expected so far. Furthermore, it has been demonstrated that B19V, similar to parvoviruses infecting animals, has a high rate of evolutionary changes that is more typical of RNA viruses (López-Bueno et al., 2006; Shackelton et al., 2005; Shackelton & Holmes, 2006). Since the NS1 gene evolves at a similar rate as the gene for VP2, it was suggested that immune selection is not the primary cause of the high substitution rate in B19V.
In the cases of sequence heterogeneity reported here, a certain similarity of polymorphism was, however, revealed. Thus, we would speculate that, in the situation presented here, a certain kind of mutational or selective mechanism is underlying. Since we only detected the polymorphism in specimens hosting two genotypes, and we and others have not observed a population of different genomes in singly infected individuals, it is tempting to speculate that polymorphism is an effect of double infection. Presuming that infections were acquired sequentially, one might speculate that the incoming infection might exert an effect on the viral genome already resident. Alternatively, given the recently described genetic instability of the virus (Shackelton & Holmes, 2006) and the fact that genotype 2 virus has widely disappeared from circulation but its genome can be frequently detected in tissue from people born before 
1960 (Norja et al., 2006), possibly a more attractive speculation is that the ‘new’ genotype 1 virus has evolved from the genotype 2 virus under immune pressure raised by infections with genotype 2 virus. In this context, it is noteworthy to mention that in earlier studies in the Aleutian mink disease parvovirus system more than one viral genome sequence was observed in highly virulent isolates (Gottschalck et al., 1991).
Genotype 2 genome 32.2 revealed a higher genetic divergence from the genotype 2 prototype strains than the other genotype 2 genomes of this study or database sequences, resulting in phylogenetic tree analysis with a separate branch within the cluster of genotype 2 genomes. This might indicate that the degree of genetic diversity among the genotype 2 viruses is similar to that observed among genotypes 3 and 1 viruses (Toan et al., 2006; Parsyan et al., 2007).
In the study presented here, B19V genotype 3 DNA was detected much less frequently than genotype 1 or 2 DNA. This difference is most likely due to the geographical distribution of genotype 3. Up to now, no genotype 3 virus was detected in Germany and all individuals of this study infected by genotype 3 were from foreign countries. The three singly infected genotype 3 individuals were from Morocco, Turkey (Istanbul) and Egypt (near the Sudanese border), respectively. The individual infected with genotypes 1 and 3 was from the Turkish Aegean coast. Thus, since it is probable that infection was not acquired in Germany, the results might indicate that genotype 3 is, or was, present in North Africa and the Near East. Of the 28 individuals singly infected by genotype 1, five individuals were not from Germany. Two individuals were from the Turkish Mediterranean coast, one from Belgium, one from the Netherlands and one from Afghanistan. Of the 23 individuals singly infected by genotype 2 one was from Turkey and one from North Yugoslavia. The singly infected genotype 1 individuals were born between 1923 and 1981, the singly infected genotype 2 individuals, however, between 1916 and 1956, with the exception of one individual being born in 1963, and the singly infected genotype 3 individuals between 1939 and 1974. The four individuals with the double infection were born between 1934 and 1947. Tissue viral loads were lower for genotype 2 than for genotype 1 (median values: genotype 2, 2.7 IU µg–1 DNA; genotype 1, 24.6 IU µg–1 DNA, determined as described by Hokynar et al., 2004, qPCR-2), which might be in line with the age restriction of genotype 2 sequences.
Not surprisingly, the B19V DNA sequences detected in the liver and bone marrow of the same individual were identical. Although the number of specimens investigated was rather small, the results demonstrate that B19V DNA persistence can take place at multiple body sites.
In summary, the results described here show that persistence of two B19V genotypes can occur in the same tissue and that there is evidence for persistence of a repertoire of genomic variants belonging to the same genotype. Finally, genotype 3 appears to be, or was, located in North Africa and in the Near East.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anderson, M. J., Higgins, P. G., Davis, L. R., Willman, J. S., Jones, S. E., Kidd, I. M., Pattison, J. R. & Tyrrell, D. A. J. (1985). Experimental parvoviral infection in humans. J Infect Dis 152, 257–265.[Medline]
Blümel, J., Eis-Hübinger, A. M., Stühler, A., Bönsch, C., Gessner, M. & Löwer, J. (2005). Characterization of parvovirus B19 genotype 2 in KU812Ep6 cells. J Virol 79, 14197–14206.
Broliden, K., Tolfvenstam, T. & Norbeck, O. (2006). Clinical aspects of parvovirus B19 infection. J Intern Med 260, 285–304.[CrossRef][Medline]
Brown, T., Anand, A., Ritchie, L. D., Clewley, J. P. & Reid, T. M. S. (1984). Intrauterine parvovirus infection associated with hydrops fetalis. Lancet 2, 1033–1034.[Medline]
Candotti, D., Etiz, N., Parsyan, A. & Allain, J.-P. (2004). Identification and characterization of persistent human erythrovirus infection in blood donor samples. J Virol 78, 12169–12178.
Cassinotti, P., Burtonboy, G., Fopp, M. & Siegl, G. (1997). Evidence for persistence of human parvovirus B19 DNA in bone marrow. J Med Virol 53, 229–232.[CrossRef][Medline]
Clewley, J. P. (1984). Biochemical characterization of a human parvovirus. J Gen Virol 65, 241–245.
Cohen, B. J., Gandhi, J. & Clewley, J. P. (2006). Genetic variants of parvovirus B19 identified in the United Kingdom: implications for diagnostic testing. J Clin Virol 36, 152–155.[CrossRef][Medline]
Corcoran, A. & Doyle, S. (2004). Advances in the biology, diagnosis and host-pathogen interactions of parvovirus B19. J Med Microbiol 53, 459–475.
Corcoran, A., Mahon, B. P. & Doyle, S. (2004). B cell memory is directed toward conformational epitopes of parvovirus B19 capsid proteins and the unique region of VP1. J Infect Dis 189, 1873–1880.[CrossRef][Medline]
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. J Virol 60, 548–557.
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.[CrossRef][Medline]
Eis-Hübinger, A. M., Reber, U., Abdul-Nour, T., Glatzel, U., Lauschke, H. & Pütz, U. (2001). Evidence for persistence of parvovirus B19 DNA in livers of adults. J Med Virol 65, 395–401.[CrossRef][Medline]
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Gallinella, G., Venturoli, S., Manaresi, E., Musiani, M. & Zerbini, M. (2003). B19 virus genome diversity: epidemiological and clinical correlations. J Clin Virol 28, 1–13.[CrossRef][Medline]
Gottschalck, E., Alexandersen, S., Cohn, A., Poulsen, L. A., Bloom, M. E. & Aasted, B. (1991). Nucleotide sequence analysis of Aleutian mink disease parvovirus shows that multiple virus types are present in infected mink. J Virol 65, 4378–4386.
Heegaard, E. D. & Brown, K. E. (2002). Human parvovirus B19. Clin Microbiol Rev 15, 485–505.
Hemauer, A., von Poblotzki, A., Gigler, A., Cassinotti, P., Siegl, G., Wolf, H. & Modrow, S. (1996). Sequence variability among different parvovirus B19 isolates. J Gen Virol 77, 1781–1785.
Hokynar, K., Söderlund-Venermo, M., Pesonen, M., Ranki, A., Kiviluoto, O., Partio, E. K. & Hedman, K. (2002). A new parvovirus genotype persistent in human skin. Virology 302, 224–228.[CrossRef][Medline]
Hokynar, K., Norja, P., Laitinen, H., Palomäki, P., Garbarg-Chenon, A., Ranki, A., Hedman, K. & Söderlund-Venermo, M. (2004). Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests. J Clin Microbiol 42, 2013–2019.
Kerr, J. R., Bracewell, J., Laing, I., Mattey, D. L., Bernstein, R. M., Bruce, I. N. & Tyrrell, D. A. (2002). Chronic fatigue syndrome and arthralgia following parvovirus B19 infection. J Rheumatol 29, 595–602.[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 2, 1159–1162.[Medline]
Kurtzman, G. J., Cohen, B. J., Field, A. M., Oseas, R., Blaese, R. M. & Young, N. S. (1989). Immune response to B19 parvovirus and an antibody defect in persistent viral infection. J Clin Invest 84, 1114–1123.[Medline]
Lehmann, H. W., Knöll, A., Küster, R. M. & Modrow, S. (2003). Frequent infection with a viral pathogen, parvovirus B19, in rheumatic diseases of childhood. Arthritis Rheum 48, 1631–1638.[CrossRef][Medline]
Liefeldt, L., Plentz, A., Klempa, B., Kershaw, O., Endres, A.-S., Raab, U., Neumayer, H.-H., Meisel, H. & Modrow, S. (2005). Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient analyzed by real-time PCR for simultaneous detection of genotypes 1 to 3. J Med Virol 75, 161–169.[CrossRef][Medline]
López-Bueno, A., Mateu, M. G. & Almendral, J. M. (2003). High mutant frequency in populations of a DNA virus allows evasion from antibody therapy in an immunodeficient host. J Virol 77, 2701–2708.
López-Bueno, A., Villarreal, L. P. & Almendral, J. M. (2006). Parvovirus variation for disease: a difference with RNA viruses? Curr Top Microbiol Immunol 299, 349–370.[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. J Clin Microbiol 37, 2483–2487.
Nguyen, Q. T., Wong, S., Heegaard, E. D. & Brown, K. E. (2002). Identification and characterization of a second novel human erythrovirus variant, A6. Virology 301, 374–380.[CrossRef][Medline]
Norja, P., Hokynar, K., Aaltonen, L.-M., Chen, R., Ranki, A., Partio, E. K., Kiviluoto, O., Davidkin, I., Leivo, T. & other authors (2006). Bioportfolio: lifelong persistence of variant and prototypic erythrovirus DNA genomes in human tissue. Proc Natl Acad Sci U S A 103, 7450–7453.
Ozawa, K., Ayub, J., Yu-Shu, H., Kurtzman, G., Shimada, T. & Young, N. (1987). Novel transcription map for the B19 (human) pathogenic parvovirus. J Virol 61, 2395–2406.
Parsyan, A., Szmaragd, C., Allain, J.-P. & Candotti, D. (2007). Identification and genetic diversity of two human parvovirus B19 genotype 3 subtypes. J Gen Virol 88, 428–431.
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 1, 664–665.[Medline]
Plentz, A., Hahn, J., Knöll, A., Holler, E., Jilg, W. & Modrow, S. (2005). Exposure of hematologic patients to parvovirus B19 as a contaminant of blood cell preparations and blood products. Transfusion 45, 1811–1815.[CrossRef][Medline]
Public Health Laboratory Service (1990). Prospective study of human parvovirus (B19) infection in pregnancy. Public Health Laboratory Service Working Party on Fifth Disease. BMJ 300, 1166–1170.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sanabani, S., Neto, W. K., Pereira, J. & Sabino, E. C. (2006). Sequence variability of human erythroviruses present in bone marrow of Brazilian patients with various parvovirus B19-related hematological symptoms. J Clin Microbiol 44, 604–606.
Schneider, B., Becker, M., Brackmann, H.-H. & Eis-Hübinger, A. M. (2004). Contamination of coagulation factor concentrates with human parvovirus B19 genotype 1 and 2. Thromb Haemost 92, 838–845.[Medline]
Servant, A., Laperche, S., Lallemand, F., Marinho, V., De Saint Maur, G., Meritet, J. F. & Garbarg-Chenon, A. (2002). Genetic diversity within human erythroviruses: identification of three genotypes. J Virol 76, 9124–9134.
Shackelton, L. A. & Holmes, E. C. (2006). Phylogenetic evidence for the rapid evolution of human B19 erythrovirus. J Virol 80, 3666–3669.
Shackelton, L. A., Parrish, C. R., Truyen, U. & Holmes, E. C. (2005). High rate of viral evolution associated with the emergence of carnivore parvovirus. Proc Natl Acad Sci U S A 102, 379–384.
Skjöldebrand-Sparre, L., Tolfvenstam, T., Papadogiannakis, N., Wahren, B., Broliden, K. & Nyman, M. (2000). Parvovirus B19 infection: association with third-trimester intrauterine fetal death. Br J Obstet Gynaecol 107, 476–480.
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.[CrossRef][Medline]
Toan, N. L., Duechting, A., Kremsner, P. G., Song, L. H., Ebinger, M., Aberle, S., Binh, V. Q., Duy, D. N., Torresi, J. & other authors (2006). Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients. J Gen Virol 87, 2941–2949.
Wong, S., Young, N. S. & Brown, K. E. (2003). Prevalence of parvovirus B19 in liver tissue: no association with fulminant hepatitis or hepatitis-associated aplastic anemia. J Infect Dis 187, 1581–1586.[CrossRef][Medline]
Woolf, A. D., Campion, G. V., Chishick, A., Wise, S., Cohen, B. J., Klouda, P. T., Caul, O. & Dieppe, P. A. (1989). Clinical manifestations of human parvovirus B19 in adults. Arch Intern Med 149, 1153–1156.[Abstract]
Young, N. S. & Brown, K. E. (2004). Parvovirus B19. N Engl J Med 350, 586–597.
Received 3 April 2007;
accepted 31 August 2007.
This article has been cited by other articles:
|
|
 |
|
  P. Norja, A. M. Eis-Hubinger, M. Soderlund-Venermo, K. Hedman, and P. Simmonds Rapid Sequence Change and Geographical Spread of Human Parvovirus B19: Comparison of B19 Virus Evolution in Acute and Persistent Infections J. Virol., July 1, 2008; 82(13): 6427 - 6433. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
