Journal of General Virology (2008), 89, 164–176
DOI 10.1099/vir.0.83053-0
Simultaneous persistence of multiple genome variants of human parvovirus B19 Beate Schneider,1 Andrea Ho¨ne,1 Rene´ H. Tolba,2 Hans-Peter Fischer,3 Johannes Blu¨mel4 and Anna M. Eis-Hu¨binger1 Correspondence Anna Maria Eis-Hu¨binger
[email protected]
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
Received 3 April 2007 Accepted 31 August 2007
The species human parvovirus B19 (B19V) can be divided into three genotypes. In this study, we addressed the question as to whether infection of an individual is restricted to one genotype. As viral DNA is detectable in tissue for long times after acute infection, we examined 87 liver specimens from adults for the presence of B19V DNA. Fifty-nine samples were found to be positive, 32 of them for genotype 1, 27 for genotype 2 and four for genotype 3. In four samples, DNA of two genotypes was detected; samples from three individuals were positive for genotypes 1 and 2 and a sample from one individual was positive for genotypes 1 and 3. Surprisingly, significant sequence heterogeneity was observed at approximately 1 % of the nucleotides of the genotype 1 genomes from individuals with double genotype 1 and 2 infection. Controls using different enzymes for genome amplification and dilutions of the template verified that nucleotide heterogeneity was due to the presence of three or more genome variants of genotype 1. In summary, the evidence shows that individuals can be infected with two different genotypes, and B19V DNA can persist as a population of different genomes. The results may have implications for the understanding of the antiviral immune response and the development of vaccines against B19V.
INTRODUCTION Human parvovirus B19 (B19V), a small non-enveloped virus, is a common pathogen typically causing a mild febrile illness with a rash during childhood (Anderson et al., 1983). 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 lifethreatening 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; Skjo¨ldebrandSparre 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 & 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 DQ4083012DQ408305 and DQ3334262DQ333428, 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.
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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) and D91.1 (GenBank accession no. AY083234) (Parsyan et al., 2007). 0008-3053 G 2008 SGM Printed in Great Britain
Persistence of genomic variants of human parvovirus B19
Until recently, genotype 2 viraemic individuals were found relatively infrequently (Blu¨mel 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-Hu¨binger 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 Specimens. A total of 87 samples of liver tissue was investigated.
Sixty-three specimens were from the patient’s own liver removed during liver transplantation, three were from the transplanted liver, five from non-transplanted liver patients and 16 specimens from autopsied individuals. From six autopsied individuals, bone marrow specimens were additionally investigated. Adequate sera, obtained before tissue collection, were available from 71 individuals. None of the individuals had received blood or blood products during the last 6 months. All specimens were obtained for diagnostic purposes. 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-Hu¨binger et al., 2001). Nested PCR for the detection of genotype 1 DNA was performed as described earlier (Eis-Hu¨binger 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 (59– 39, positions according to GenBank accession no. AX003421): 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 uC. Positive
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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 (EisHu¨binger 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, DV and VP1/VP2 (Hemauer et al., 1996), DV-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, DVG2, 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 DV-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 DV-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 IM81 (Blu¨mel et al., 2005) using the primer pair DV-IIIG2 [forward, nt 2123–2142 (59–39, positions according to GenBank accession no. AY044266); 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 pDVIIIG1 and pDV-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 165
B. Schneider and others 10210 (approx. 3.26109–3.2 copies ml21) of plasmid pDV-VIIIG1 were prepared in a nucleic acid eluate obtained from a B19V DNAnegative liver specimen. Each plasmid (1 ml) pD-VIIIG1 dilution was mixed with 1 ml of plasmid pDV-IIIG2 solution held at constant concentration (approx. 3.26109 copies ml21) and subjected to nested PCR using primer set DV-IIIG1. Except for a template volume of 2 ml 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 ¢46104 genome equivalents per reaction as determined by plasmid titration.
RESULTS Detection of more than one B19V genotype in single individuals A total of 87 liver specimens was investigated by screening PCRs for genotypes 1, 2 and 3 DNA, respectively, followed by verification of the positive results by direct nucleotide analysis of a large B19V genome region (Fig. 1). 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 DVG1/DV-IIIG1 (the latter one for two specimens for which no amplicons could be generated by DV) and the primer sets NS1-CG2 and DVG2, 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). Overall, genotypes 1, 2 and 3 DNA was detected in 32, 27 and four specimens, respectively. All sequences were either
typical genotype 1, 2 or 3 sequences (Fig. 2). 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. Genetic diversity within the B19V genotypes Direct sequence analysis of the genotype 1 genomes derived from the genotypes 1 and 2 co-infected specimens (sequences 31.1, 32.1, 33.1 from specimens 31, 32, 33, respectively) reproducibly revealed nucleotide heterogeneity at single positions. To verify this result, direct sequence analysis of the three genomes was extended to nt (Au) 158– 5047 representing 87 % of the B19V genome. At least four independently amplified PCR products of each target region were sequenced in both directions using the primer sets given in Supplementary Table S2 (available in JGV Online). 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
Fig. 1. Schematic diagram showing the genome regions of B19V amplified for tissue screening and DNA sequencing. The length of the viral genome is depicted at the top. The numbers below the bars indicate the nucleotide positions according to the genotype 1 prototype strain Au (GenBank accession no. M13178). For genotype 3 genomes, the nucleotide numbering is according to genotype 3 subtype D91.1 (GenBank accession no. AY083234), and for better comparison, the numbering according to Au is given in parentheses. The genome fragments amplified by the screening PCRs are given including the inner primers, the sequenced genome regions are given without the primers. For genomes sequenced over approximately 1 kb, the primer sets used are given above the bars. For the largely sequenced genomes, the primers sets used are given in Supplementary Tables S2– S4 (available in JGV Online). 166
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Fig. 2. A phylogenetic tree constructed by the neighbour-joining method based on the genomic region between nt 1940 and 3020 (positions according to genotype 1 prototype strain Au, GenBank accession no. M13178). The DNA isolates presented in this study are numbered consecutively and are compared with the prototype strains for genotypes 1, 2 and 3, given in bold (GenBank accession nos: Wi, M24682; LaLi, AY044266; A6, AY064475; V9, AX003421; and D91.1, AY083234). For the calculation of the divergence of sequences 31.1, 32.1 and 33.1, the sequence with maximum divergence to genotype 1 prototype strain Au was used.
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). Overall, the nucleotide polymorphisms were identified at a total of 105 genomic positions. Notably, at 26 and 21 of these positions the type of heterogeneity was identical in two and three genomes, respectively. Additionally, at position 1392, heterogeneity (C/A) was identical in two genomes, while the third genome displayed three bases (A/ T/C) including the two bases detected in the other DNA isolates. Similarly, at position 924 one genome displayed C/ T, while another one displayed A/T/C. Thus, 47 % (49/105) of the polymorphic positions were identical in at least two genomes. Furthermore, in nearly all of these positions the type of heterogeneity was identical. http://vir.sgmjournals.org
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 167
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Table 1. Sequence analysis of the genotype 1 genomes with nucleotide polymorphisms (sequences 31.1, 32.1 and 33.1) The sequenced region spanned nt (Au) 158–5047. Shown are all positions that differ from genotype 1 prototype strain Au (GenBank accession no. M13178). For further comparison, the nucleotides at the respective positions of 25 long sequenced genotype 1 genomes available in the database are given. The GenBank accession numbers of these sequences are AY386330 (J35), AF162273 (HV), AY504945 (NAN), M24682 (Wi), Z68146 (Stu), AB030693 (Mi), AB030694 (Rm), AB030673 (N8), AB126262 (AN23), DQ225149 (SN807), DQ225148 (KyMa), DQ225151 (AnTo), DQ225150 (OsFr), DQ293995 (C39), Z70560 (I/1), Z70528 (2/II), AF113323, AJ781031 (1), AJ781032 (2), AJ781033 (3), AJ781034 (4), AJ781035 (5), AJ781036 (6), AJ781037 (7) and AJ781038 (8). n, Number of sequences available at the indicated position. Deduced amino acid exchanges refer to those nucleotides that differ from the nucleotides of prototype strain Au. Nucleotide heterogeneities are indicated in bold. Nucleotide Nucleotide in Au position in Au
Promoter region 223 245 272 307 310 313 318 333 337 343 354 430 NS1 480 597 649 692 723 750 783 787 795 859 888 915 924 972 1018 1035 1050 1089 1173 1275 1296 1321 1392 1476 1530 1569 1591 1608 1769 1773 1873 2093
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Genotype 1 genomes
Amino acid exchange
Nucleotides in additional genotype 1 genomes
31.1
32.1
33.1
n
T T T C G C G T G G C C
T/G – C/T – A/G – G/C C/A A/G – C/T A
T/G C/T C/T C/A – G/C – C/A – – – A
T/G – C/T – A/G – G/C C/A A/G G/C C/T A
8 10 10 10 10 10 10 10 11 11 11 18
8 10 2 10 10 8 10 7 11 11 11 18
T T T/8 C C G C/1 A/1 T G C/3 A G G C A
A G G C T T G C T T A T C C T A T T C C T T T A A T G A C A A C
G A/G – T C – A/G C/T C/T – A/G C/T C/T – T/G A/T C/T – – – – C/T C/A – A/G A/T – A/G – – – –
G – A/G T C C/T – – – – A/G C/T – C/T T/G A/T C/T C/T C/T C/A C/T C/T A/T/C C/A A/G A/T – – C/A A/G – C/T
G A/G – T C – A/G C/T C/T C/T A/G C/T A/T/C – T/G A/T C/T – – – – C/T C/A – A/G A/T A/G A/G – – A/G –
25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25
25 25 25 25 11 15 17 25 24 24 25 25 24 25 25 25 25 25 24 25 25 25 25 25 18 21 25 25 25 25 25 25
G G G T A/11 C/3 T T/10 C A/8 G C T/1 C T/1 C A T C/1 A C T A T T C/1 T C T T C A G/7 A T/3 C/1 A G A C A A C
EAK TAI
SAA
DAE
AAT TAN IAV SAL
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Table 1. cont. Nucleotide Nucleotide in Au position in Au
2096 2223 2226 2268 2283 2329 2352 2400 2433 VP1u 2453 2527 2530 2531 2560 2618 2689 2711 2786 2962 2987 3013 3022 3047 3063 VP2 3172 3174 3175 3182 3187 3223 3247 3264 3292 3307 3334 3355 3380 3508 3511 3583 3610 3625 3691 3757 3809 3923 3988 3994 4000 4106 4249 4261 http://vir.sgmjournals.org
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Amino acid exchange
31.1
32.1
33.1
T T A G C G A C C
– – – C – – A/G C/T –
C/T C/T A/G C C/T A/G – C/T C/T
– C/T – C – – A/G C/T –
A A G G A C A C T A A A A G C
G A/G A/G G/C A/G T A/G – – A/G – A/G A/G – C/T
G A/G A/G – – C/T – C/T – A/G A/G – – A/G –
G G C T C T C C G G G C C T G A G G A T T T C A A T A A
– – G/C C C/T C – – – A A/G T – – A/G A/G – A/G A/G C/T A C/T – C/A C/A C/T C/A A/G
A/G A/G G/C C C/T C – C/T A/G A A/G T C/T – A/G A/G T/G A/G A/G – A – C/T – – – – –
G A/G A/G G/C A/G C/T A/G – C/T A/G – – – – – – – – G/C C C/T C/T C/T – – A A/G T – C/T A/G A/G – A A/G C A C – C/A C/A C/T C/A A/G
Nucleotides in additional genotype 1 genomes n
FAS
EAK
KAE
VAL
HAY
IAV
AAT AAV
GAD SAP
PAL
PAS
SAT
25 25 25 25 25 25 25 25 25
23 25 25 25 25 25 17 24 24
T/2 C T A C C G G/8 A C/1 T C/1 T
25 25 25 25 25 25 25 25 25 25 25 25 25 25 25
25 23 25 15 25 24 25 25 25 24 25 25 25 25 25
G A/2 G G G/10 C A C/1 T A C T A/1 G A A A G C
25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25
21 25 25 24 13 25 22 25 24 23 25 25 25 25 24 23 25 22 24 25 25 25 13 25 24 24 25 23
G/4 T G C C/1 T C/11 T/1 G C C/3 T C G/1 A A/2 T G T C T G/1 A A/2 G G G/3 A A/1 G T A T T/12 C A A/1C T/1 C A A/2 G 169
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Table 1. cont. Nucleotide Nucleotide in Au position in Au
4265 4342 4349 4399 4462 4498 4537 4570 4591 4606 4624 4660 4699 4720 4841 4859 4893 4908 4915 4940
C G C G A A A C T G G G C A A G A G T A
Genotype 1 genomes
Amino acid exchange
Nucleotides in additional genotype 1 genomes
31.1
32.1
33.1
n
C/T – – – – – – T – – A A/G – – – – – – – –
– A/G – A/G – A/G – T – – A/G – C/T – – T/G C/A A/G A/T –
C/T – C/T – A/T A/G A/G T C/T A/G A/G A/G C/T A/G A/G – – – – A/G
25 25 25 25 25 25 25 25 25 25 25 25 25 25 24 21 13 13 13 11
25 25 25 25 25 23 25 20 22 25 24 24 24 25 24 21 13 13 13 11
C G C G A A/2 G A C/5 T T/3 C G G/1 A G/1 T C/1 T A A G A G T A
positions represented by T or C in the comparison strains except for one position where one comparison strain displayed G.
NSNIG2, NSNIIG2, VP1/VP2G2, VPintG2, VPCG2 and VPextG2 (all genomes), FinG2 (genomes 31.2 and 32.2), and ProG2 (genome 31.2), respectively.
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 nonsynonymous 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).
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.
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
Genetic diversity among the genotypes within a patient
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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. Journal of General Virology 89
Persistence of genomic variants of human parvovirus B19
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 coamplification 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 DV-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 (n572) where the reference strains Au and LaLi and the genomes 31.1 and 31.2 were not homologous to each other. For further verification of sequence polymorphism, dilutions of the DNA preparation of specimen 31 were subjected to PCR with primer set DV-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. http://vir.sgmjournals.org
To exclude the possibility that the detected polymorphism of genotype 1 sequences was an artificial consequence of the presence of genotype 2 DNA in the same specimen, experiments with mixtures of cloned genotype 1 and 2 DNAs were performed. Homologous genome regions were amplified from plasmid pGEM-1/B19 and from genotype 2 virus IM-81 using the outer primers of primer set DV-IIIG1 and primers DV-IIIG2, respectively. Equal volumes of serially 10-fold diluted plasmid pDV-IIIG1 (approx. 3.26109–3.2 copies) and constant amounts of plasmid pDV-IIIG2 (approx. 3.26109 copies) were mixed and subjected to first and second round PCR using the primer set DV-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 1024 of pDV-IIIG1 (approx. 3.26106 copies) were unambiguously genotype 1. At pDV-IIIG1 dilutions 1025–10210 (approx. 3.26105–3.2 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. Fiftynine 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 171
B. Schneider and others
Fig. 3. Sequence analysis of the polymorphic genotype 1 genome 31.1 after amplification with four different enzymes. With enzymes A, B, C and D eight (A1–A8), four (B1–B4 and C1–C4) and one (D1) independent PCR and sequencing reactions were performed, respectively. The sequenced region spanned nt (Au) 2247–3284. Given are all nucleotides that are not identical between the 31.1 sequence, genotype 1 reference strain Au (GenBank accession no. M13178), genotype 2 reference strain LaLi (GenBank accession no. AY044266) and genotype 2 genome 31.2 at the corresponding positions. Sequences are listed according to their similarity. Nucleotides that differ between the reference strains Au and LaLi are highlighted in yellow and blue, respectively. Nucleotides in the genomes 31.1 and 31.2 are coloured accordingly. Dark green background indicates nucleotides that are identical between the reference strains and the genomes 31.1 or 31.2. Light green background indicates nucleotides that differ from the nucleotides depicted in dark green. Ambiguities are shown as colourless.
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Journal of General Virology 89
Persistence of genomic variants of human parvovirus B19
Table 2. Identification of three types of unambiguous genotype 1 DNA sequences from genotypes 1 and 2 co-infected specimen, 31, by PCR using diluted template Sequenced region, nt (Au; GenBank accession no. M13178) 2245–3285. Nucleotide position in Au
Nucleotide in Au
2268 2352 2400 2453 2527 2530 2531 2560 2618 2689 2962 3013 3063 3175 3182 3187 3223 Sequence type
G A C A A G G A C A A A C C T C T
Dilution 1:2
1:4
1:5
1 : 32
C* A C G A A G G T G A G T C C C C III
C A C G A A G G T G A G T C C C C III
C G T G G G G A T A G A C G C T C I
C A C G A A C G T G A A C C C C C IV
*Shown are positions that differ from genotype 1 prototype strain Au.
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 DV or NS1-CG2 and DVG2, respectively.
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 Blu¨mel 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
DISCUSSION Long-term persistence of B19V DNA in human tissue is a well known feature (Cassinotti et al., 1997; Eis-Hu¨binger et al., 2001; So¨derlund 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 http://vir.sgmjournals.org
Table 3. Genotypes 1 and 2 DNA in bone marrow and liver specimens from the same individual Specimen
16 40 56 Neg 1 Neg 2 Neg 3
Anti-B19 IgG antibody
+ + 2 NSAd + 2
Bone marrow
Liver
Genotype*
Genotype*
1
2
3
1
2
3
+D 2 2 2 2 2
2 +D +D + 2 2
2 2 2 2 2 2
+D 2 2 2 2 2
2 +D +D 2 2 2
2 2 2 2 2 2
*Sequenced region spanned nt (Au; GenBank accession no. M13178) 1912–3023. DSequences within one individual were identical. dNo serum available. 173
B. Schneider and others
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 (Lo´pez-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 (Lo´pez-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 174
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, Journal of General Virology 89
Persistence of genomic variants of human parvovirus B19
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 mg21 DNA; genotype 1, 24.6 IU mg21 DNA, determined as described by Hokynar et al., 2004, qPCR2), which might be in line with the age restriction of genotype 2 sequences.
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. 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.
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.
Deiss, V., Tratschin, J. D., Weitz, M. & Siegl, G. (1990). Cloning of the
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.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.
ACKNOWLEDGEMENTS The technical assistance of Ulrike Reber is highly acknowledged. We thank Bernd Kupfer for the valuable discussions. This work was supported by the Commission of the European Community (Grant QLK2-CT-2001-00877) and by BONFOR (Grant 0-151.0021).
human parvovirus B19 genome and structural analysis of its palindromic termini. Virology 175, 247–254. Eis-Hu¨binger, A. M., Reber, U., Abdul-Nour, T., Glatzel, U., Lauschke, H. & Pu¨tz, U. (2001). Evidence for persistence of
parvovirus B19 DNA in livers of adults. J Med Virol 65, 395–401.
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. 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.
REFERENCES
Hokynar, K., So¨derlund-Venermo, M., Pesonen, M., Ranki, A., Kiviluoto, O., Partio, E. K. & Hedman, K. (2002). A new parvovirus
Anderson, M. J., Jones, S. E., Fisher-Hoch, S. P., Lewis, E., Hall, S. M., Bartlett, C. L. R., Cohen, B. J., Mortimer, P. P. & Pereira, M. S. (1983).
Hokynar, K., Norja, P., Laitinen, H., Paloma¨ki, P., Garbarg-Chenon, A., Ranki, A., Hedman, K. & So¨derlund-Venermo, M. (2004).
Human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet 1, 1378.
Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests. J Clin Microbiol 42, 2013–2019.
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. Blu¨mel, J., Eis-Hu¨binger, A. M., Stu¨hler, A., Bo¨nsch, C., Gessner, M. & Lo¨wer, 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. Brown, T., Anand, A., Ritchie, L. D., Clewley, J. P. & Reid, T. M. S. (1984). Intrauterine parvovirus infection associated with hydrops
genotype persistent in human skin. Virology 302, 224–228.
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. 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. Kurtzman, G. J., Cohen, B. J., Field, A. M., Oseas, R., Blaese, R. M. & Young, N. S. (1989). Immune response to B19 parvovirus and
fetalis. Lancet 2, 1033–1034.
an antibody defect in persistent viral infection. J Clin Invest 84, 1114–1123.
Candotti, D., Etiz, N., Parsyan, A. & Allain, J.-P. (2004). Identification
Lehmann, H. W., Kno¨ll, A., Ku¨ster, R. M. & Modrow, S. (2003).
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.
Frequent infection with a viral pathogen, parvovirus B19, in rheumatic diseases of childhood. Arthritis Rheum 48, 1631–1638. 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. J Gen Virol 65, 241–245.
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.
Cohen, B. J., Gandhi, J. & Clewley, J. P. (2006). Genetic variants of
Lo´pez-Bueno, A., Mateu, M. G. & Almendral, J. M. (2003). High
parvovirus B19 identified in the United Kingdom: implications for diagnostic testing. J Clin Virol 36, 152–155.
mutant frequency in populations of a DNA virus allows evasion from antibody therapy in an immunodeficient host. J Virol 77, 2701–2708.
Clewley, J. P. (1984). Biochemical characterization of a human
http://vir.sgmjournals.org
175
B. Schneider and others Lo´pez-Bueno, A., Villarreal, L. P. & Almendral, J. M. (2006).
Sanabani, S., Neto, W. K., Pereira, J. & Sabino, E. C. (2006). Sequence
Parvovirus variation for disease: a difference with RNA viruses? Curr Top Microbiol Immunol 299, 349–370.
variability of human erythroviruses present in bone marrow of Brazilian patients with various parvovirus B19-related hematological symptoms. J Clin Microbiol 44, 604–606.
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.
Schneider, B., Becker, M., Brackmann, H.-H. & Eis-Hu¨binger, A. M. (2004). Contamination of coagulation factor concentrates with
Nguyen, Q. T., Sifer, C., Schneider, V., Allaume, X., Servant, A., Bernaudin, F., Auguste, V. & Garbarg-Chenon, A. (1999). Novel
human parvovirus B19 genotype 1 and 2. Thromb Haemost 92, 838–845.
human erythrovirus associated with transient aplastic anemia. J Clin Microbiol 37, 2483–2487.
Servant, A., Laperche, S., Lallemand, F., Marinho, V., De Saint Maur, G., Meritet, J. F. & Garbarg-Chenon, A. (2002). Genetic diversity within
Nguyen, Q. T., Wong, S., Heegaard, E. D. & Brown, K. E. (2002).
human erythroviruses: identification of three genotypes. J Virol 76, 9124–9134.
Identification and characterization of a second novel human erythrovirus variant, A6. Virology 301, 374–380.
Shackelton, L. A. & Holmes, E. C. (2006). Phylogenetic evidence for
Norja, P., Hokynar, K., Aaltonen, L.-M., Chen, R., Ranki, A., Partio, E. K., Kiviluoto, O., Davidkin, I., Leivo, T. & other authors (2006).
Shackelton, L. A., Parrish, C. R., Truyen, U. & Holmes, E. C. (2005).
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. Plentz, A., Hahn, J., Kno¨ll, 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. 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. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
176
the rapid evolution of human B19 erythrovirus. J Virol 80, 3666–3669. High rate of viral evolution associated with the emergence of carnivore parvovirus. Proc Natl Acad Sci U S A 102, 379–384. Skjo¨ldebrand-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. So¨derlund, 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. 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. 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. Young, N. S. & Brown, K. E. (2004). Parvovirus B19. N Engl J Med
350, 586–597.
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