Human gyrovirus (HGyV) is a recent addition to
the list of agents found in humans. Prevalence, biologic
properties, and clinical associations of this novel virus are
still incompletely understood. We used qualitative PCRs
to detect HGyV in blood samples of 301 persons from
Italy. HGyV genome was detected in 3 of 100 solid organ
transplant recipients and in 1 HIV-infected person. The virus
was not detected in plasma samples from healthy persons.
Furthermore, during observation, persons for whom
longitudinal plasma samples were obtained had transient
and scattered presence of circulating HGyV. Sequencing
of a 138-bp fragment showed nucleotide identity among all
the HGyV isolates. These results show that HGyV can be
present in the blood of infected persons. Additional studies
are needed to investigate possible clinical implications.
gyrovirus (HGyV) (1). The characteristics of its genome—a
single, closed molecule of circular, negative-sense DNA
≈2,300 nt long—and sequence homology with the chicken
anemia virus (CAV) have suggested that HGyV might be
the fi rst human-infecting member of the genus Gyrovirus,
which is part of the family Circoviridae and encompasses
only 1 previously known species, CAV (2).
The genome of HGyV, which resembles CAV more
closely than other members of the family (1,3), contains
an untranslated region of ≈380 nt and 3 major partially
overlapping open reading frames (named viral protein [VP]
1, VP2, and VP3) that encode proteins of 465, 231, and
31 aa, respectively. Whether HGyV and equivalent CAV
proteins have similar functions is unknown. VP3 products
n 2011, Sauvage et al. reported the discovery of a novel
virus in human skin specimens and named it human
of HGyV and CAV (for which the coded protein has been
named apoptin) share short, functionally pivotal amino acid
motifs, suggesting that HGyV also encodes an apoptin-like
protein. The CAV apoptin induces tumor-specifi c apoptosis
in a p53-independent fashion and has been shown to be a
potential anticancer therapeutic agent in various animal
The epidemiology, biologic properties, and pathogenic
potential of HGyV remain poorly understood. Sauvage
et al. (1) detected the HGyV genome in nonlesional skin
specimens of healthy persons and 1 HIV-positive patient
but not in respiratory and fecal samples. This observation
suggests that HGyV is most likely part of the normal skin
microfl ora of humans, similarly to other recently discovered
viruses (9,10). However, like related animal viruses, CAV
infects a large range of cell types and causes a variety of
pathologies (including bone marrow aplasia leading to
aplastic anemia, hemorrhage, and lymphoid depletion) and
increased death in young chicken (11). Also, CAV infection
has been associated with the worsening of pathologies
caused by other viral and bacterial agents (11–13).
Thus, because HGyV might cause clinically relevant
disorders, guidance in choosing the directions for clinical
investigation is crucial and needs to come from studies
aimed at defi ning the prevalence of HGyV infection in
different human populations, portal of entry, type of cells
targeted during primary amplifi cation, and site of latency/
persistence. We investigated the presence of HGyV DNA in
blood samples of 301 persons in Italy using specifi c PCRs.
The results indicated overall HGyV positivity of 1.3%.
Materials and Methods
Patients and Samples
During December 2011, we studied 301 randomly
selected persons living in central Italy. Most (251) were
diseased patients whose blood samples had been submitted
to the Virology Unit, Pisa University Hospital (Pisa,
Human Gyrovirus DNA in
Human Blood, Italy
Fabrizio Maggi, Lisa Macera, Daniele Focosi, Maria Linda Vatteroni, Ugo Boggi, Guido Antonelli,
Marc Eloit, and Mauro Pistello
956 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 6, June 2012
Author affi liations: Pisa University Hospital, Pisa, Italy (F. Maggi,
L. Macera, D. Focosi, M.L. Vatteroni, U. Boggi, M. Pistello);
“Sapienza” University of Rome, Rome, Italy (G. Antonelli); Institut
Pasteur, Paris, France (M. Eloit); and Pathoquest, Paris (M. Eloit)
Human Gyrovirus, Italy
Italy), by local hospitals for routine virologic analysis;
the remaining 50 were healthy blood donors. The patients
comprised 151 HIV-infected persons (mean ± SD age 47
± 14 years [range 18–80 years]; 115 men) who, before
initiation of the study, had received no antiretroviral
treatment. The patients also were examined for xenotropic
murine-leukemia virus-related virus in a previous study
(14). The other 100 patients (mean ± SD age 56 ± 8 years
[range 36–69 years]; 71 men) were solid organ transplant
recipients: 50 had received a liver transplant, and 50
had received a kidney transplant. Plasma samples were
collected from patients on the day of transplant and then
at selected times after transplant. Aliquots were prepared
immediately, stored, and kept under sterile conditions at
–80°C until use. Written informed consent was collected
from each patient.
HGyV DNA Detection
Viral DNA was extracted from 400 μL of peripheral
whole blood or 200 μL of plasma by using the Maxwell
16 System (Promega, Madison, WI, USA) or QIAamp
DNA blood kit (QIAGEN, Hilden, Germany), respectively,
according to the manufacturers’ instructions. Extracted
DNA was amplifi ed with 2 PCR protocols (developed and
provided by V. Sauvage et al.), which target the VP1 gene
of the viral genome.
The fi rst amplifi cation was a single-step TaqMan
real-time PCR (rtPCR) designed on a 72-nt fragment.
The assay was performed in a 25-μL volume
containing 400 nmol/L of each primer (HGyV-rtFP:
5′-CCCTGCAAGTGCTGAGGATAA-3′), 200 nmol/L
double-labeled probe (HGyV-rtP: 5′-FAM-CAAAGAGC
extracted viral DNA, and the Universal PCR Master
Mix (Applied Biosystems, Foster City, CA, USA)
containing deoxynucleoside triphosphate, and Taq DNA
polymerase. Reaction was run in triplicate for each sample
in an iQCYCLER rtPCR detection instrument (Bio-Rad
Laboratories, Hercules, CA, USA) by using a standardized
program (95°C 10 min; 45 cycles of 15 s at 95°C, and 60°C
60 s; and 40°C 30 s). A sample was considered rtPCR
positive when HGyV DNA was detected in 2 of 3 replicas
and when amplifi cations were specifi c as determined by
2% agarose gel electrophoresis.
A 178-bp fragment was amplifi ed by a nested PCR
format, described by Sauvage et al. (1) with modifi cations.
This PCR was performed for 25 cycles with sense primer
HGyV-OF (5′-CAAAATCGGAGGCCCTAACCC-3′) and
antisense primer HGyV-OR (5′-ATGCCTGAATAGCTGC
CAGCC-3′) under the following conditions: denaturation
at 94°C for 60 s, annealing at 55°C for 60 s, and extension
at 72°C for 45 s. The product of this reaction (10 μL)
was then re-amplifi ed for 35 cycles with internal primers
HGyV-IF (5′-GGTCAGCACAAACGACGCAG-3′) and
HGyV-IR (5′- AGGTCTCCCATAGCGTCCAG-3′) at the
same PCR conditions. The reactions were conducted in
a 50-μL PCR mixture containing Taq DNA polymerase,
each deoxynucleoside triphosphate at a concentration of 10
mmol/L, primers (20 μmol/L each), and optimized buffer
All samples were tested at least in duplicate and on
different occasions. The amplifi ed product was analyzed by
electrophoresis on a 2% agarose gel after ethidium bromide
staining. Amplicon size was compared with standard
molecular mass markers. To minimize contamination risk,
serum handling, DNA extraction, PCR amplifi cation, and
electrophoresis analysis were conducted in separated rooms.
Negative controls were added during DNA extraction and
PCR amplifi cation. To validate the amplifi cation process,
positive controls (obtained from M.E.) were run in each
All HGyV PCR–positive isolates were characterized
by sequencing a 138-bp fragment (from nt 1328 to 1465
of VP1 gene of the representative isolate FR823283)
encompassing the target region of nested PCR. PCR
amplicons, purifi ed from the gel by using a QIAquick
Gel Extraction Kit (QIAGEN, Chatsworth, CA, USA),
were sequenced by using the Big Dye Terminator Cycle
Sequencing Kit (Applied Biosystems) and an automatic
DNA sequencer (ABI model 3130; Applied Biosystems).
Nucleotide sequences were aligned with the only sequence
available at GenBank at the time of writing and by using
the ClustalW algorithm included in BioEdit version 220.127.116.11
Evidence of HGyV Infections
No samples from 50 healthy blood donors studied
yielded positive results for HGyV (Table 1). Of 4 samples
in which HGyV DNA was detected, 3 (6%) were from
kidney transplant recipients and 1 (0.7%) was from an
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 6, June 2012 957
Table 1. Prevalence of human gyrovirus DNA in 251 HIV-positive
or transplant recipient patients and 50 blood donors, Italy
Healthy persons Plasma
HIV-positive patients Whole blood
Organ transplant recipients
No. (%) HGyV
Longitudinal Study of HGyV Viremia in
We examined plasma samples from 100 transplant
patients for whom we had sequential samples obtained at
selected times after transplant. Three of these patients who
had received a kidney transplant tested positive for HGyV
DNA, indicating that they had systemic HGyV infection.
When additional samples of these patients were examined,
a similar pattern emerged (Table 2). Plasma samples
from 2 transplant recipients were already HGyV positive
when they were fi rst examined before transplantation.
Subsequently, HGyV DNA detection was intermittent in
the posttransplant samples: it was positive at month 12
(patient AL) and 6 (patient CV) but negative in the other
samples tested. For patient MG, we examined 4 blood
samples obtained before and after HGyV detection in
plasma. At all these times, the plasma tested repetitively
negative for HGyV DNA.
Genetic Analysis of HGyV Isolates
Sequencing was conducted on all 6 PCR fragments
obtained. All the isolates were related to the previously
published strain, and the sequences obtained were virtually
identical in the nucleotide fragment examined (online
Appendix Figure, wwwnc.cdc.gov/EID/article/18/6/12-
0179-FA1.htm). When blood specimens from the same
patient were sequenced, no nucleotide change was noted
among the viral sequence fragments obtained at any time.
The recent discovery of a human virus similar to
CAV prompted an investigation of samples collected
from patients and from healthy blood donors in central
Italy. This investigation confi rms that HGyV is present in
humans, extends the previous fi ndings, and raises several
points. In the only study published, the HGyV genome had
been investigated in 115 nonlesional skin specimens from
adults and 138 specimens (46 nasopharyngeal aspirates and
92 fecal samples) from children. HGyV DNA was found
in only 5 nonlesional skin specimens (1), suggesting that
the virus could be a member of the human skin virome.
In our study, plasma samples were taken from 251
immunocompromised patients (151 patients with HIV
infection and 100 transplant recipients) and 50 healthy
donors. HGyV DNA was demonstrated in the plasma of 4
persons, all with dysfunctional immune systems.
The presence of HGyV in blood of infected humans
suggests that the infection might also be systemic. The
fi nding is not totally unexpected because CAV and the
recently discovered avian gyrovirus 2, a virus genetically
similar to HGyV, can circulate in the blood of infected
animals (15–18). The clinical signifi cance of HGyV
viremia and relationship to induction of pathogenic
processes is unclear. However, among the patients in
whom virus was demonstrated, most had received a kidney
transplant and thus had severe underlying nephropathy.
The 3 kidney transplant recipients were a 52-year-old
man with focal segmental glomerulosclerosis, a 21-year-
old woman with lacrimoauriculodentodigital syndrome
(i.e., Levy-Hollister syndrome), and a 57-year-old man
with end-stage renal failure of unknown cause. All 3
recipients received basiliximab induction and triple
maintenance immunosuppressive therapy with prednisone,
mycophenolate mofetil, and cyclosporine A. The remaining
HGyV-positive patient was a 32-year-old HIV-positive
man who, when tested for HGyV, had an HIV load of
156,000 copies/mL and a CD4+ count of 465 cells/μL.
Data collected over time showed the occasional
detection of viral DNA in blood. In fact, a similar pattern
was observed in all the positive patients studied: plasma
HGyV-positive samples alternated with virus-negative
samples, indicating that circulating virus was intermittent.
This fi nding also was confi rmed in the HIV-positive patient,
for whom the only additional plasma sample obtained 18
months after HGyV detection was HGyV negative (data
not shown). Analysis of more data from additional studies
is needed to understand the role of this transient detection
of the virus, which might represent a putative short-lived
acute infection with possible subsequent re-infection or
just declines of the HGyV load under the lower limit of
sensitivity of the detection methods used.
The limited size of the PCR fragment sequenced
does not enable us to determine with certainty whether
the HGyV DNA detected before transplantation and at
later times were the same. This information could explain
possible reinfections and/or persistence of the virus. Further
molecular studies with larger fragments from variable
regions of HGyV genome will be necessary to evaluate
whether the virus persists in the infected host.
Dr Maggi is a clinical virologist at the Clinical Virology
Unit of the Pisa University Hospital. His main interest is in
natural history and immunopathogenesis of infection by viruses,
particularly emerging viruses.
958 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 6, June 2012
Table 2. Time course of human gyrovirus DNA detection in 3
kidney transplant patients, Italy*
transplant Patient AL
*HGyV, human gyrovirus; ND, not determined.
†The urine sample tested at month 12 was HGyV negative.
Plasma HGyV DNA
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Address for correspondence: Fabrizio Maggi, Virology Unit, Pisa
University Hospital, Via San Zeno 37, I-56127 Pisa, Italy; email: fabrizio.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 6, June 2012 959