JOURNAL OF VIROLOGY, Aug. 2011, p. 7948–7950
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 15
Identification of the First Human Gyrovirus, a Virus Related
to Chicken Anemia Virus?†
Virginie Sauvage,1Justine Cheval,2Vincent Foulongne,3Meriadeg Ar Gouilh,1Kevin Pariente,1
Jean Claude Manuguerra,1Jennifer Richardson,4Olivier Dereure,5Marc Lecuit,6
Ana Burguiere,1Vale ´rie Caro,7and Marc Eloit2,4,8*
Institut Pasteur, Laboratory for Urgent Responses to Biological Threats, 25 rue du Docteur Roux, F-75015 Paris, France1; Pathoquest,
28 rue du Docteur Roux, F-75015 Paris, France2; Laboratoire de Virologie, CHU de Montpellier, St. Eloi, 80 Avenue A. Fliche,
34295 Montpellier, France3; Ecole Nationale Ve ´te ´rinaire d’Alfort, UMR 1161 Virologie ENVA, INRA, ANSES,
7 avenue Ge ´ne ´ral de Gaulle, F-94704 Maisons Alfort, France4; Service de Dermatologie, CHU de Montpellier,
St. Eloi, 80 Avenue A. Fliche, 34295 Montpellier, France5; Institut Pasteur, Inserm, Microbes and
host barriers Group, 28 rue du Docteur Roux, F-75015 Paris, France6; Institut Pasteur, Genotyping of
Pathogens and Public Health Platform, 28 rue du Docteur Roux, F-75015 Paris, France7; and
Institut Pasteur, Department of Virology, 28 rue du Docteur Roux, F-75015 Paris, France8
Received 30 March 2011/Accepted 18 May 2011
We have identified in a skin swab sample from a healthy donor a new virus that we have named human
gyrovirus (HGyV) because of its similarity to the chicken anemia virus (CAV), the only previously known
member of the Gyrovirus genus. In particular, this virus encodes a homolog of the CAV apoptin, a protein that
selectively induces apoptosis in cancer cells. By PCR screening, HGyV was found in 5 of 115 other nonlesional
skin specimens but in 0 of 92 bronchoalveolar lavages or nasopharyngeal aspirates and in 0 of 92 fecal samples.
The chicken anemia virus (CAV) is highly contagious and
causes severe anemia, hemorrhage, and depletion of lymphoid
tissue through the destruction of bone marrow progenitor cells
in young chickens. CAV has been the only known species of
the Gyrovirus genus, which is part of the family Circoviridae.
CAV is a nonenveloped virus, with an icosahedral capsid 19 to
27 nm in diameter surrounding a single-stranded (ss) circular
genomic DNA (8). In a skin swab taken from a healthy person,
we discovered a new human virus that we have designated
human gyrovirus (HGyV) because of its homology with the
chicken anemia virus.
DNA was extracted with an automatic EasyMag apparatus
(bioMe ´rieux, Marcy l’Etoile, France) and amplified using the
bacteriophage phi29 polymerase-based multiple-displacement
amplification (MDA) assay and random primers. The reaction
was performed using a Repli-g Midi kit (Qiagen, Courtaboeuf,
was conducted using an Illumina HighSeq 2000 sequencer.
High-molecular-weight DNA resulting from isothermal am-
plification was fragmented into 200- to 350-nucleotide (nt)
fragments, to which adapters were ligated. A large number
(7,588,712) of reads of 100 nt each were obtained from the
sample. Illumina sequences were first sorted by a subtractive
database comparison procedure as previously described (2). In
short, the whole human genome sequence (NCBI build 37.1/
assembly hg19) was scanned with SOAPaligner (http://soap
.genomics.org.cn). A very restrictive BLASTN study was also
performed to eliminate additional host reads. The CLC
Genomics Workbench program was used for de novo assembly.
The comparison of the results of analysis of the single reads
and contigs to known genomic and taxonomic data was per-
formed using dedicated specialized viral and generalist data-
bases maintained locally (i.e., the entire NCBI nucleotide da-
tabase or only the extracted viral part). The aforementioned
databases were scanned using BLASTN and BLASTX. We
used Paracel BLAST software, which distributes the BLAST
tasks among multiple processors. Taxonomic assignments were
based on the lowest common ancestor from the best hits
among reads with a significant e-value (generally 10-e4).
Among different contigs harboring a size compatible with a
viral genome, a contig of 2,315 nt assembled from 436 reads
was more deeply analyzed, as it disclosed similarities to CAV.
Although CAV from chickens with severe anemia was de-
scribed and isolated more than 30 years ago (10), no virus of
this genus is currently known in humans, and this finding was
Based on the sequence of this contig, we defined a set of
primers allowing amplification of the entire genome (see Table
S1 in the supplemental material) and then confirmed the se-
quence by the Sanger method. The resulting sequence of the
virus we have named HGyV (Human Gyrovirus) (accession
number FR823283), along with its principal features, is pre-
consists of a single molecule of circular, single-stranded nega-
tive-sense DNA that forms a closed circle. The complete ge-
nome of CAV is 2,290 to 2,320 nucleotides long and contains
three partially overlapping open reading frames encoding
three proteins, VP1, VP2, and VP3. The genome of HGyV is
2,315 nt long and displays similar characteristics (Fig. 1; see
also Fig. S1 in the supplemental material). Alignment of the
whole genome of HGyV with that of CAV is depicted in Fig.
* Corresponding author. Mailing address: Department of Virology,
28 rue du Docteur Roux, F-75015 Paris, France. Phone: 33 1 44389216.
Fax: 33 1 40 61 39 40. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 1 June 2011.
S1 in the supplemental material. The overall nucleotide iden-
tity between the two genomes is low: in the region of maximal
identity, between nt 100 and 700 (CAV accession number
M55918), the overall identity is around 70%.
The genome of HGyV presents an overall organization sim-
ilar to that of CAV. It contains a 5? region displaying general
features of the CAV promoter-enhancer (6) (see Fig. 2). In
particular, the 5? region of HGyV comprises repeated ele-
ments of 22 nt (ATGT….TCA C/T C/A), designated direct
repeat a (DRa) to DRe. The DR elements of HGyV and CAV
contain the sequences ACGTCA and AGCTCA, respectively,
which both differ by a single nucleotide from the estrogen-
responsive element (ERE) half-site (A)GGTCA. In HGyV,
there is also a less-conserved repetition just upstream of DRa
(C/A G/T GTACAGGGGGGTACGTCA T/C C/A at posi-
tions nt 153 to 174; note that this sequence also contains the
ACGTCA sequence [not shown in Fig. 2]). In HGyV, these
DRs are repeated without intervening sequences. In CAV, 4 to
5 (depending on the strain) copies of these repeats are present
and are separated by linkers of 15 nt between the first two or
three elements and the last two. While we did not find any
evidence of the presence of the ERE found in the vicinity of nt
50 in CAV, we identified a palindromic sequence (CAATCA
GAATTG) downstream from DRe that shows a certain simi-
larity to it. Indeed, while EREs generally consist of pentamers
or hexamers such as (A)GGTCA separated by a 2- to 3-nt
insert, this consensus sequence has been shown to occur in a
wide range of variations (1).
Like CAV, the HGyV genome contains three partially over-
lapping open reading frames encoding different proteins. We
have used the names of the CAV counterparts to identify those
open reading frames (see details in Table S2 in the supple-
mental material). The CAV VP1 is a 51-kDa capsid protein
that also contains motifs for rolling-circle replication in the C-ter-
minal region. Given the distance from the other ss DNA viruses,
phylogenetic analysis was impossible. CAV VP3, a nonstructural
protein, is also called apoptin. The alignment of HGyV apoptin
with CAV apoptin is shown in Fig. 3, together with the domains
of CAV apoptin that have been shown to be functionally impor-
tant (see review in reference 4). The overall identity of HGyV
apoptin with CAV apoptin is relatively low (31%).
The CAV apoptin alone can induce apoptosis in a broad
range of cancer cells but not in nontransformed or primary
cells (4). It induces apoptosis by a mechanism implicating the
mitochondrial (intrinsic) pathway and is thus independent of
the death receptor (extrinsic) pathway. As shown in Fig. 3, the
so-called N-terminal leucine-rich stretch (LRS), characterized
by a high content of hydrophobic aliphatic leucine or isoleu-
cine residues, seems conserved. This region interacts with dif-
ferent cellular proteins that seem important for its function
FIG. 1. Map of HGyV (accession number FR823283) and principal
features. Principal features are designated by homology with cognate
features in the annotated sequence of CAV (accession number M55918).
FIG. 2. Nucleotide sequence of the promoter region of HGyV and principal features (see text). Direct repeats (DRa to DRe) of 22 nt are shown
with putative estrogen-responsive elements (ACGTCA; underlined). The palindromic CAAT CAGA ATTG putative estrogen-responsive element
and the TATA box are depicted.
VOL. 85, 2011NOTES 7949
(summarized in reference 4). A bipartite nuclear localization Download full-text
signal (NLS), comprising motifs NLS1 and NLS2, also seems
to be conserved, together with the putative nuclear exportation
signal (NES). Of note, the Netphos 2.0 server predicts a phos-
phorylation site at position 111 of HGyV apoptin, which is
located between NES and NLS2 and seems homologous to
position Thr-108 of CAV apoptin. In fact, nuclear accumula-
tion of CAV apoptin, which is important for its proapoptotic
activity, is dependent on the phosphorylation of Thr-108. This
phosphorylation is mediated by a kinase (cyclin-dependent ki-
nase 2) that can be constitutively active in tumors and trans-
formed cells but not in normal cells (5). Apoptin expression is
able to induce cell cycle arrest and apoptosis in p53 null cells
(4) and is a very attractive candidate for use in cancer therapy.
Nevertheless, CAV apoptin is derived from a virus restricted to
birds. It can therefore be anticipated that HGyV apoptin
would be more potent in mammals than its avian counterpart.
Since this new virus was first identified in a skin swab sample,
we have investigated HGyV prevalence in a set of 115 nonle-
sional skin specimens sampled by cutaneous swabbing from
volunteer subjects by a nested-PCR assay targeting the VP1
gene (see Table S3 in the supplemental material). Consent was
obtained for collection of human samples according to French
regulations. Subjects included 35 patients hospitalized or at-
tending outpatient clinics at the dermatology unit of the Mont-
pellier university hospital, 20 immunocompromized patients
(including 10 HIV-1-infected patients and 10 renal transplant
recipients), and 60 healthy control subjects enrolled within the
Montpellier university hospital staff. Since respiratory and fe-
cal-oral routes of transmission could be involved in the epide-
miology of the virus, we also tested additional sets of human
samples, including 46 bronchoalveolar lavage (BAL) fluid sam-
ples from intensive care unit patients and 46 nasopharyngeal
aspirates plus 92 fecal samples from children attending the
pediatric unit of the Montpellier university hospital. Caution
was exercised to limit the risk of cross-contamination. HGyV
was detected in five additional skin samples, and specificity was
confirmed by sequencing the amplicons. Four of these HGyV-
positive samples were recovered from healthy volunteers and one
from an HIV-positive patient. We failed to detect HGyV se-
quences in both respiratory and stool samples. Our results show
that HGyV is a putative member of the human skin virome
detected in nearly 5% of individuals and is reminiscent of its close
relative CAV, which is transmitted by contaminated feathers (3).
The importance of HGyV could be 2-fold. First, the poten-
tial association of HGyV with disease needs to be determined.
Also, the description of a HGyV apoptin provides a promising
lead for development of biotherapy for cancer in human and
animals (7). It may also serve as a tool to decipher pathways of
apoptosis in cancer cells. We are currently exploring these
different aspects of HGyV biology.
Nucleotide sequence accession number. The sequence of
HGyV has been deposited in the NCBI database under acces-
sion number FR823283.
The platform “Genotyping of Pathogens and Public Health” is sup-
ported in part by the Institut de Veille Sanitaire (Saint-Maurice,
France). This study was partly supported by grants from both region Ile
de France and the Programme Hospitalier de Recherche Clinique of
the Montpellier University Hospital (AOI 2008, Protocole UF8425).
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FIG. 3. Comparison of the primary structures of CAV and HGyV apoptin. HGyV apoptin (H-apoptin) was aligned with its CAV homolog
(A-apoptin) (accession number P54094) by the use of the CLC Genomics Workbench program. Principal domains of the CAV apoptin protein
are shown (LRS, leucine-rich domain; NLS1 and NLS2, nuclear localization signals; NES, putative nuclear exportation signal). The amino acid Thr
in position 108 of CAV apoptin is depicted by an arrow. The phosphorylation site at amino acid Thr-111 of HGyV apoptin predicted by the
Netphos program (www.cbs.dtu.dk/services/NetPhos/) is also shown.
7950 NOTESJ. VIROL.