J. Clin. Microbiol.
Véronique Auguste and Antoine Garbarg-Chenon
Xavier Allaume, Annabelle Servant, Françoise Bernaudin,
Quang Tri Nguyen, Christophe Sifer, Véronique Schneider,
Transient Aplastic Anemia
Novel Human Erythrovirus Associated with
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JOURNAL OF CLINICAL MICROBIOLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Aug. 1999, p. 2483–2487 Vol. 37, No. 8
Novel Human Erythrovirus Associated with Transient
QUANG TRI NGUYEN,1* CHRISTOPHE SIFER,1VE´RONIQUE SCHNEIDER,2XAVIER ALLAUME,1
ANNABELLE SERVANT,1FRANC ¸OISE BERNAUDIN,3VE´RONIQUE AUGUSTE,1
AND ANTOINE GARBARG-CHENON1
Laboratoire de Virologie, Ho ˆpital Armand Trousseau (EA 2391 UFR Saint-Antoine),1and Laboratoire de Virologie,
Ho ˆpital Rothschild (EA 2391 UFR Saint-Antoine),275 571 Paris Cedex 12, and Service de Pe ´diatrie,
Centre Hospitalier Intercommunal de Cre ´teil, 94 010 Cre ´teil Cedex,3France
Received 21 December 1998/Returned for modification 15 April 1999/Accepted 6 May 1999
Erythrovirus (formerly parvovirus) B19 causes a wide range of diseases in humans, including anemia due to
aplastic crisis. Diagnosis of B19 infection relies on serology and the detection of viral DNA by PCR. These
techniques are usually thought to detect all erythrovirus field isolates, since the B19 genome is known to
undergo few genetic variations. We have detected an erythrovirus (V9) markedly different from B19 in the
serum and bone marrow of a child with transient aplastic anemia. The B19 PCR assay yielded a product that
hybridized only very weakly to the B19-specific probe and whose sequence diverged more from those of 24 B19
viruses (11 to 14%) than the divergence found within the B19 group (<6.65%). Restriction enzyme analysis of
the V9 genome revealed that this genetic divergence extended beyond the amplified region. Interestingly,
serological tests failed to demonstrate a response characteristic of acute B19 infection. V9 could be a new
erythrovirus, and new diagnostic tests are needed for its detection.
Erythrovirus B19, called parvovirus before the revision of
the taxonomy in 1995 (17), causes erythema infectiosum, usu-
ally in children and young adults. In most patients B19 infec-
tion causes an acute illness from which patients recover spon-
taneously and confers protective, lifelong immunity (12, 27).
Complications can occur when the viral infection arises in
patients with particular backgrounds: chronic thrombocytope-
nia or anemia in immunocompromised patients and fetal in-
fection in pregnant women. Transient aplastic crisis (TAC), a
frequent complication of acute B19 infection, was originally
described as the abrupt onset of severe anemia with reticu-
lopenia in patients suffering from chronic hemolysis due to
cessation of erythrocyte production in the bone marrow sec-
ondary to the tropism of the virus for erythroid progenitor
cells. TAC can also occur under conditions of erythroid stress,
such as hemorrhage or iron deficiency (5, 27). Erythrovirus
B19 is a common infectious agent in humans: B19 seropreva-
lence is approximately 50% by the age of 15 and rises further
among elderly people because infection occurs throughout
adult life (5). Although it is generally accepted that B19 infec-
tion is transmitted by the respiratory route, it can also be
transmitted by blood or blood products, even those treated
with heat or a solvent-detergent to inactivate viruses (5).
Genotypes were previously established on the basis of the
restriction enzyme polymorphism of the viral genome (15,
16). However, until now, the single-stranded DNA of eryth-
rovirus has been known to undergo little genetic variation
(?1% of the entire genome [4, 11, 22]), and there is only
one species in the genus Erythrovirus, which is referred to as
MATERIALS AND METHODS
B19 serological assays. Testing for B19-specific antibodies was performed with
commercial assays (Parvovirus B19 IgG Enzyme Immunoassay or Parvovirus B19
IgM Enzyme Immunoassay; Biotrin, Dublin, Ireland), according to the manu-
Viral DNA extraction. To isolate leukocytes, the bone marrow sample was
layered onto Histopaque 1119 (Sigma Diagnostics, Saint Quentin-Fallavier,
France) according to the manufacturer’s instructions. After two washes in 150
mM NaCl, the leukocytes were pelleted by centrifugation and stored at ?80°C.
The cells were lysed in 250 ?l of buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl,
2.5 mM MgCl2, 0.5% Tween 20, 0.5% Nonidet P-40) with 15 ?g of proteinase K
at 56°C for 90 min. After inactivation of the proteinase K by heating at 100°C for
10 min, the solution was extracted with 250 ?l of phenol and then 250 ?l of
chloroform-isoamyl alcohol (25/1). The DNA was precipitated at ?80°C for 1 h
with 20 ?g of glycogen (as the carrier), 25 ?l of 3 M sodium acetate (pH 5.2), and
500 ?l of ethanol. After washing with 70% ethanol, the DNA pellet was dried at
56°C for 10 min, resuspended in 100 ?l of H2O, and stored at ?20°C.
Viral DNA was extracted from serum samples with the QIAamp Blood Kit
(Qiagen, Courtabœuf, France), according to the manufacturer’s instructions, and
was stored at ?20°C.
Restriction map. NaCl was added to the (single-stranded) viral DNA extracted
from the serum sample to a final concentration of 50 mM. The solution was
heated at 95°C for 2 min and annealed at 55°C for 16 h. The double-stranded
DNA was digested with a restriction enzyme (BamHI, HindIII, or PvuII).
PCR for B19 detection. A standard PCR procedure was performed with 10 ?l
of either the extracted DNA or a 1/10 dilution of it with the following program:
1 cycle of 95°C for 7 min; 40 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C
for 1 min; and 1 cycle of 72°C for 7 min (on a thermocycler 480, Perkin-Elmer,
Courtabœuf, France). The PCR products were separated by electrophoresis on
a 1.5% agarose gel and Southern transferred by capillary blotting (0.4 M NaOH,
0.6 M NaCl) onto a charged nylon membrane. The blot was hybridized at 42°C
for 16 h in a buffer (50% formamide, 5? SSC [1? SSC is 0.15 M NaCl plus 0.015
M sodium citrate], 2% blocking reagent [Boehringer, Mannheim, Germany],
0.1% N-laurylsarcosine, 0.02% sodium dodecyl sulfate) with a B19-specific
probe, P2560, labeled with the Dig Oligonucleotide Tailing Kit (Boehringer)
according to the manufacturer’s instructions. Posthybridization washes were
done at 60°C, and detection was performed with alkaline phosphatase conjugate
and CSPD (DIG Luminescent Detection Kit; Boehringer). Sequences corre-
sponding to the primers and probe are located in the unique region of the VP1
gene (VP1u) of the B19 sequence (22): primer 376 from positions 2408 to 2428,
primer 377 from positions 2790 to 2809 (in the reverse orientation), and probe
P2560 from positions 2560 to 2600 (13).
Sequencing of PCR products. Both strands of the PCR products from the bone
marrow and serum extracts were sequenced with primers 376 and 377 by using
the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer Applied
Biosystems, Courtabœuf, France) and the ABI Prism 377 DNA Sequencer (Per-
kin-Elmer Applied Biosystems).
* Corresponding author. Mailing address: Institut Pasteur, Unite ´ de
Ge ´ne ´tique et Biochimie du De ´veloppement, 25 rue du Dr. Roux, 75
724 Paris Cedex 15, France. Phone: 33 1 45 68 85 65. Fax: 33 1 40 61
34 40. E-mail: email@example.com.
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Sequence analysis and phylogeny. The sequence of the PCR product was used
as the query sequence in FASTA (19) and BLAST (1) searches of the GenBank
and EMBL data banks with the GCG package (Genetics Computer Group,
The 24 nonidentical data bank B19 sequences covering 346 bp of the amplified
region were aligned with the V9 sequence by using CLUSTAL W or CLUSTAL
X (25). Phylogenetic analyses were performed with the PHYLIP package (9):
Nucleotide distances were estimated with DNADIST, and the phylogenetic tree
was then calculated with NEIGHBOR. The phylogenetic tree was plotted by
using TREE TOOL (14).
Nucleotide sequence accession numbers. The EMBL nucleotide sequence
accession no. of the sequences reported here are AJ223617 and AJ242810.
Clinical and biological findings. A 6-year-old male child
who had lived in France since his birth was admitted to a
pediatric unit with headache and dysuria in May 1995. Clinical
examination noted only paleness of the conjunctiva and a mild
fever (temperature, 38.6°C). Initial blood analyses (Table 1)
revealed severe microcytic anemia with a sharp drop in reticu-
locytes associated with lymphopenia and neutropenia (5 May
1995). The myelogram (12 May 1995) showed a relative rich-
ness in erythroblasts and few abnormalities: insufficient hemo-
globin in erythroblasts and negative Perls’ staining for iron. No
giant pronormoblasts were found. Complementary analyses
indicated that numerous factors could have contributed to this
anemia. Gastrofibroscopy conducted because of initially low
serum iron levels (5.7 ?mol/liter; normal range, 11 to 24 ?mol/
liter) revealed mild interstitial gastritis associated with Helico-
bacter pylori in biopsy specimens of the antrum. Three months
after the initial crisis, investigation of the chronic hemolysis
(haptoglobin concentration, ?0.09 g/liter) revealed a glucose-
6-phosphate dehydrogenase (G6PD) defect (on 8 August 1995
tests for G6PD revealed 0 U per g of hemoglobin; normal
range, 5.3 to 7.9 U per g of hemoglobin). A familial inquiry
found a G6PD defect in one of his four siblings.
The anemia evolved favorably over a few weeks with treat-
ment of the sideropenia and the gastritis (ferrous fumarate,
folic acid, omeprazol, amoxicillin, and clarithromycin). No
transfusion or gamma globulin injection was given.
Virological findings. A search for a B19 infection was con-
ducted. B19 serology was negative for immunoglobulin M
(IgM) and positive for IgG both initially (on 2 May 1995 the
optical density and cutoff values were 0.030 and 0.090 respec-
tively, for IgM and 0.473 and 0.376, respectively, for IgG) and
3 months later (on 7 August 1995 the optical density and cutoff
values were 0.012 and 0.082 respectively, for IgM and 1.130
and 0.279, respectively, for IgG). Despite the serological re-
sults which suggested a past B19 infection, a bone marrow
sample was taken on 12 May 1995 for a PCR search for the B19
viral genome. This B19 PCR was inconclusive (Fig. 1), giving
contrasting results: a PCR band that migrated the same dis-
tance as the B19-positive control band was clearly visible on
the electrophoresis gel stained with ethidium bromide, but this
band gave only a very faint signal after hybridization of the
Southern blot with our B19-specific probe (Fig. 1). The inten-
sity of the hybridization signal was enhanced when less strin-
gent washing conditions were used (data not shown). This
finding strongly suggests a mispairing of the B19-specific probe
with an erythrovirus PCR product but could also denote non-
specific hybridization of the probe to a PCR product unre-
lated to B19 (false-positive result). Similar results were ob-
tained when the B19 PCR was done with the first serum
(drawn on 2 May). Conversely, the PCR assay was negative
with the serum sample drawn 3 months later (data not
TABLE 1. Hematological and biological data
3 May 1995
5 May 1995
13 May 1995
22 May 1995
aND, not done.
2484 NGUYEN ET AL.J. CLIN. MICROBIOL.
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Sequence analysis and phylogeny. To determine whether
this weak hybridization signal could be due to mutations in the
probe recognition sequence, the PCR products obtained from
both the bone marrow and the serum DNA extracts were
sequenced. Both sequences were identical, with six mutations
mismatching the 41-bp B19-specific probe, and this weak sim-
ilarity (85%) could explain the very faint hybridization signal
that was observed (Fig. 1).
When compared to sequences stored in GenBank and
EMBL databases, this 346-bp sequence resembled B19 se-
quences of the VP1u gene by FASTA analysis (data not
shown). However, the sequence from this novel erythrovirus
(which we called V9) was unexpectedly more divergent from
the sequences of 24 B19 isolates (?11.07% divergence) (Fig.
2) than these B19 sequences are among themselves (?6.65%
divergence). The unrooted phylogenetic tree based on these
data showed that the V9 sequence was outside the B19 group
(Fig. 3). Figure 3 would have been even more striking and
the B19 cluster would have been even more condensed if we
had excluded sequence 24 (pvb19x556), which was obtained
from a patient with a persistent B19 infection (10), which is
quite unusual in a nonimmunocompromised patient (5, 7,
To determine whether V9 could be derived from an animal
parvovirus, all known animal parvovirus sequences were sub-
jected to phylogenetic analysis. The V9 sequence was situated
very far from the animal parvovirus sequences, as is the B19
sequence (6). Among animal parvovirus sequences, the simian
parvovirus was the closest to both V9 and B19, with diver-
gences of 47 and 44%, respectively.
In light of these results, we rescreened samples for which,
like the V9 sample, the results of our routine B19 PCR test
were initially found to be inconclusive (work on this is in
progress). From the first eight samples drawn from patients in
France in early 1998, one, named R1, gave a PCR product
whose sequence was similar to that of V9 (5.20% divergence)
and even more distant from B19 (?12.14% divergence) than
V9 is (Fig. 2 and 3). This patient suffered from macrocytic
anemia in a background of chronic renal insufficiency.
Restriction fragment length analysis. One should point out
that the amplified VP1u region was recently found to be the
most variable region of the B19 genome (8, 10), especially for
B19 strains isolated during persistent infections. In this region,
FIG. 1. B19 PCR with bone marrow extract. (A) Agarose gel stained with
ethidium bromide. (B) Southern blot hybridized to a B19-specific probe. The
arrowheads indicate the positions of the expected 402-bp PCR product. CT, B19
positive control; L, 100-bp DNA molecular size ladder; BM, bone marrow ex-
tract. The B19 PCR assay performed with the bone marrow extract gave a band
which migrated the same distance as that of the B19-positive control (A), but this
PCR product hybridized very poorly to the B19-specific probe (B). Exposure
times were identical for both sides of the figure.
FIG. 2. Multiple alignments of partial VP1u sequences from V9, R1, and the
most divergent B19 sequences. (a) DNA sequences (346 bp) Modified from
reference 18 with permission of the publisher. The genetic divergence between
the sequences of V9 and R1 strains and the sequences of B19 strains resides in
the 5? part of the VP1 gene, which corresponds to one of the two major neu-
tralization epitopes localized in the first 80 amino acids of VP1 (2, 21).
VOL. 37, 1999A NOVEL HUMAN ERYTHROVIRUS? 2485
on June 18, 2013 by Inserm IFR6
pvb19x556 shows 4% divergence with pvbaua, according to
Hemauer et al. (10), and up to 6.65% divergence when its
sequence is compared to those of the Chinese isolates. To
determine whether the sequence divergence that we observed
between V9 and B19 extended beyond the VP1u region, the
entire V9 genome isolated from serum was subjected to re-
striction enzyme analysis (Fig. 4). Undigested V9 DNA had an
apparent molecular size similar to that of B19 DNA. The V9
DNA restriction patterns obtained with the enzymes BamHI,
HindIII, and PvuII differed markedly from those of 65 B19
strains from different geographic origins reported in the liter-
ature (15, 16, 26). V9 DNA had no BamHI site and one
HindIII site, while all the B19 strains have one and two to three
sites for these enzymes, respectively. Five PvuII sites were
found for V9, whereas one to three PvuII sites were found for
B19 strains. On the basis of its restriction map, V9 cannot be
assigned to one of the five B19 genotypes described by Mori
et al. (15), nor does it seem to be related to any of the B19
strains unassigned to a given genotype. Thus, it is quite like-
ly that the sequence divergence between V9 and B19 is not
restricted to the VP1u region but extends along the whole
This case report demonstrates that human erythrovirus
genomic sequences may be much more divergent than has so
far been accepted. Sequence divergence in the VP1u region
can be due to antigenic drift within a patient during persistent
B19 infection due to selective pressure by the immune system
over a long period of viral replication (10). However, with
regard to the V9 virus, such a mechanism is unlikely for two
reasons: (i) V9 was found during an acute infection and (ii)
when compared to the B19 sequence (22), the DNA mutations
did not systematically result in nonconservative amino acid
substitutions, as has been shown for persistent B19 infections
(10). Furthermore, contrary to what was described for persis-
tent B19 infections (10), the genetic divergence between V9
and B19 seems to extend beyond the VP1u region, as indicated
by the V9 restriction map. Such a high level of divergence
raises numerous questions which must be addressed.
First, this high level of divergence can affect the diagnosis of
erythrovirus infection either by B19 PCR and DNA hybridiza-
tion assays or by B19 serological assays. The results of the B19
PCR test that we used were indeed inconclusive, and the re-
sults of B19 IgM serological assay were negative, while this
patient’s clinical and biological presentations suggested an
acute human erythrovirus infection, for which both tests are
usually positive (7, 12). Diagnostic tests specific for V9 DNA
and antibody detection are being developed to address this
point. Using these tools, large-scale epidemiological studies
will be conducted in order to evaluate the medical importance
of V9-related erythroviruses. The finding that such viruses are
widespread would require the virological diagnosis of erythro-
virus infection to be changed, thereby extending the etiological
role of the erythroviruses in human pathology.
FIG. 3. Unrooted phylogenetic tree of partial VP1u gene sequences from V9,
R1, and B19 genomes. Branch lengths are proportional to genetic distances,
expressed as percent divergence (a scale bar is shown at the bottom of the
figure). Correspondence of the numbers used in the figure and the GenBank
mnemonics for B19 sequences are as follows: 1, ebu38506; 2, ebu38507; 3,
pvb19x572; 4, bvu31358; 5, ebu38510; 6, ebu38511; 7, ebu38514; 8, pvb19x583;
9, pvb19x591; 10, ebu38508; 11, pvb19x560; 12, ebu38512; 13, ebu38546; 14,
pvbaua; 15, pvb19x528; 16, ebu38515; 17, pvbpro; 18, ebu38513; 19, pvb19nsvp;
20, pvb19x599; 21, e09420; 22, pvb19x541; 23, ebu38509; 24, pvb19x556. Eryth-
rovirus B19 sequences 1 (ebu38506) and 2 (ebu38507) were isolated from Chi-
nese patients, and B19 sequence 24 (pvb19x556) came from a patient suffering
from persistent B19 infection. V9 and R1 are clearly outside the B19 group.
FIG. 4. Restriction map of V9 genome. Restriction enzymes were used to cut
V9 DNA. Lane 2, uncut DNA; lane 3, BamHI-digested DNA; lane 4, HindIII-
digested DNA; lane 5, PvuII-digested DNA. DNA molecular size markers were
as follows: lane 1, 1-kb ladder; lane 6, 100-bp ladder (Life Technologies, Cergy
Pontoise, France). Lane 7, uncut B19 DNA.
2486 NGUYEN ET AL. J. CLIN. MICROBIOL.
on June 18, 2013 by Inserm IFR6
Second, the initial B19 IgG serological assay positivity could Download full-text
indicate a prior B19 infection or, alternatively, an (early) cross-
reactivity to the B19 serological assay. If the former hypothesis
is true, it appears (at least for this patient) that the acquired
immunity against B19 could not prevent the occurrence of an
acute infection with variant erythrovirus V9. Because a previ-
ous B19 infection usually confers strong and durable protective
immunity against reinfection with B19 (12), this case questions
whether anti-B19 antibodies ensure cross-protective immunity
against V9. Measurements of VP1 IgG avidity (23) or epitope
type-specific IgG reactivity to VP2 (24) should indicate wheth-
er this patient had preexisting B19 immunity. Should large
epidemiological studies confirm this supposition, it will be nec-
essary to include V9 proteins in the development of any can-
didate human erythrovirus vaccine (3, 12, 20). It should be
noted that in the VP1u region most of the divergence between
V9 and B19 resides in the 5? portion (Fig. 2), which corre-
sponds to one of the two major neutralization epitopes (2, 21).
Third, the taxonomic position of erythrovirus variant V9 as
a new genotype in the B19 species or a new species in the genus
Erythrovirus remains to be established. We favor the latter
hypothesis for two reasons: the V9 genome restriction pattern,
which differs from those of unassigned and established B19
genotypes reported worldwide (15, 16, 26), and the genetic
divergence between V9 and B19s. The results of phylogenetic
analysis with V9, R1, and B19 sequences reinforce the hypoth-
esis that V9 and R1 could belong to another species besides
the species B19 in the genus Erythrovirus. All B19 sequences
group in a compact cluster (?6.65% divergence), while the V9
and R1 sequences clearly segregate from this group (with di-
vergences of ?11.07 and ?12.14%, respectively) and seem to
belong to a second cluster (5.20% divergence between V9 and
R1). This hypothesis must be confirmed by phylogenetic anal-
ysis with both a wider portion of erythrovirus genome and a
larger number of variant erythrovirus sequences. Cloning and
sequencing of the whole V9 viral genome, which is already in
progress, may help to fully elucidate its taxonomic position.
Fourth, because erythrovirus variant V9 was found during a
TAC in a child and this clinical presentation is typical of a B19
infection, some questions arise: Why was V9 not found earlier?
Is it a new variant (or a new virus) that segregated very recently
from B19 or, more simply, is it an ancient human virus that has
not been detected until now because diagnostic tests (serology,
DNA hybridization, PCR) were designed for B19 and are not
suitable for the detection of V9? Epidemiological studies with
V9-specific assays may help to provide an answer. If the prev-
alence of V9 is initially low and rises over the next few years,
this may be a clue that V9 is an emerging human virus.
We thank Eric Osika, Charles Roth, and Janet Jacobson for critical
reading of the manuscript.
This work was supported in part by FRM grant 4001524-01 and
DRC AP-HP grant TBI 97029.
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