B R I E F R E P O R T
Detection of Adeno-Associated Virus
Viremia in Hematopoietic Cell
Judson Heugel,1,aMichael Boeckh,1,2Meei-Li Huang,1,2Becky Dierks,2
1Fred Hutchinson Cancer Research Center;2University of Washington, Seattle, and;
3Seattle Cancer Care Alliance, Washington
Adeno-associated virus (AAV) is widely considered to be
nonpathogenic, but the clinical epidemiology of this virus is
limited. By use of polymerase chain reaction assays, we in-
vestigated the incidence and clinical significance of AAV
viremia in a population of consecutive recipients of a
hematopoietic cell transplant (HCT). Four (2.8%) of 145
patients developed AAV viremia after HCT. Viremia was low
level and transient in all patients. Two patients were admitted
to the hospital and died in proximity to AAV viremia
(,7 weeks between diagnosis and death); however, AAV was
not detected in tissue specimens obtained at autopsy.
Thus, AAV does not appear to play a pathogenic role in
organ-specific illness, even in a highly immunocompro-
The incidence and clinical impact of wild-type adeno-associated
virus (AAV) infection among hematopoietic cell transplant
(HCT) recipients is unknown. AAV, a parvovirus, establishes
a latent infection by integrating into the host genome locus on
chromosome 19 . Replication competency can be reestab-
lished by coinfection with a helper virus , typically an
adenovirus or herpesvirus. AAV is widely considered to be
nonpathogenic in immunocompetent individuals, and re-
combinant vectors based on AAV have generated considerable
interest for genetherapies andcontemporaryvaccine
approaches. However, knowledge of the incidence and clinical
correlates of AAV viremia in immunocompromised patients has
yet to be defined. We developed an AAV-specific real-time
polymerase chain reaction (PCR) assay to investigate the spec-
trum of clinical disease associated with AAV viremia in a cohort
of HCT recipients.
Materials and Methods
The study population was an unselected, prospectively accrued
cohort of 145 pediatric and adult patients who received non–
T-cell–depleted allogeneic or autologous HCT with myeloa-
blative conditioning in 1998. Weekly plasma samples from these
patients were collected from the day of stem cell infusion until
day 100 after transplantation or until death, whichever occurred
earlier. Samples were frozen at 280?C and evaluated in batches
for evidence of AAV viremia. Patients gave informed consent,
and the study was approved by the Fred Hutchinson Cancer
Research Center institutional review board.
A quantitative PCR assay was developed to detect AAV sub-
types 1–5, 7, and 8. We modified our pan1/pan3 primer set
according to previously published sequences for AAV1, 2, 3, 5,
and 6 , and we designed specific Taqman probes for each set.
Eachprimer/probe set was thentestedonAAV1, 2,3,4,5,7,and
8. The set derived from AAV2 detected all AAV strains except
AAV3, with similar or improved efficiency than the type-specific
probes. AAV3 could be detected only with an AAV3-specific set.
Therefore, the final PCR reaction contained primers and probes
derived from AAV2 and AAV3.
DNA was extracted from 200 lL of plasma, using QIAmap96
DNA blood kits (Qiagen), and from tissue biopsy specimens,
using EZ1 tissue kits on a BioRobot EZ1 workstation (Qiagen).
was then used for each PCR reaction. Each 25-lL PCR reaction
consisted of 12.5 lL of QuantiTect multiplex PCR master mix
AAA-AAG-TCC-TTG-ACT-TCC-TGT-TT); and 100 nmol/L of
each probe (probe 1: FAM-TTG-CAA-GAC-CGG-ATG-TTC-
AAA-TTT-TAMRA; probe 2: FAM-CTG-CAG-GAC-CGG-ATG-
TTT-GAA-TTT-TAMRA). EXO internal control was spiked into
the PCR master mix to monitor for false-negative results, as de-
scribed elsewhere . The 7500 Fast Real-Time PCR system
(Applied Biosystems) was used to test PCR reactions. The PCR
cycling conditions were 95?C for 15 minutes to activate the
polymerase, followed by 45 1-minute cycles at 94?C and 1 minute
Received 14 January 2011; accepted 20 July 2011; electronically published 17 October
Presented in part: 45th Annual Meeting of the Infectious Disease Society of America, San
Diego, California, 4 October 2007.
aPresent affiliation: Department of Pediatrics, Johns Hopkins Hospital, Baltimore, Maryland.
Correspondence: Lawrence Corey, MD, Fred Hutchinson Cancer Research Center, 1100
Fairview Ave N, Seattle, WA 98109 (email@example.com).
The Journal of Infectious Diseases
? The Author 2011. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
0022-1899 (print)/1537-6613 (online)/2011/20411-0014$14.00
d JID 2011:204 (1 December)
d BRIEF REPORT
The specificity of the PCR reaction was tested on 37 strains of
adenoviruses, human genome, cytomegalovirus (CMV), Epstein
Barr virus, varicella zoster virus, human herpesvirus (HHV) 6,
HHV-8, herpes simplex virus 1, JC virus, BK virus, and parvo-
virus B-19. No cross amplification was detected. The assay de-
tected all 7 strains of AAV (1, 2, 3, 4, 5, 7, and 8) obtained from
the American Type Culture Collection and was sensitive to
detect 10 copies of AAV consistently. The specificity was
further confirmed by no detection of AAV in DNA extracted
from 10 plasma samples, 26 peripheral blood mononuclear
cell samples, and 23 saliva samples collected from healthy
Bronchoalveolar lavage (BAL) and tissue specimens obtained
at autopsy in the context of clinical care were processed in real
time for pathogenic bacterial, viral, and fungal organisms, as
described elsewhere , and residual samples were stored at
280?C. BAL and tissue specimens obtained at autopsy from
patients who had AAV detected in their plasma were tested for
AAV by quantitative PCR. BAL and lung tissue samples were
additionally tested by PCR using published protocols for fungal
pathogens [6, 7] and for 14 respiratory viruses [8, 9], including
respiratory syncytial virus, adenoviruses, influenza viruses A and
B, parainfluenza virus types 1–4, human metapneumovirus,
rhinoviruses, and all 4 non–severe acute respiratory syndrome
Clinical, laboratory, and radiographic findings were corre-
lated with AAV viremia events. All patients in our study were
evaluated previously for CMV and HHV-6 plasma viremia ,
and a subset were evaluated previously for BK virus and ade-
novirus viremia [10, 11]. These data were explored for evidence
of coinfection in our patients with AAV viremia.
Of the 145 HCT recipients, 34% received an HLA-matched,
related transplant; 6% received a mismatched, related trans-
plant; 43% received an unrelated transplant; and 17% received
an autologous transplant. Thirty-seven patients (26%) died
within 100 days after transplantation. The mean age of patients
undergoing HCT was 40.7 years; only 14 patients were younger
than 21 years. Most (58%) were men. The underlying diseases
were described elsewhere .
AAV was detected in plasma samples from 4 (2.8%) of 145
HCT recipients. The patient and virologic characteristics are
described in Table 2. Briefly, AAV was detected a mean of 47.5
days (range, 21–77 days) after transplantation. The average viral
load was ,1000 copies/mL, and the duration of viral detection
was brief, ranging from 1 to 3 weeks (median duration, 1 week).
Two of the 4 patients with AAV viremia (patients 1 and3) were
admitted to the hospital with lung infiltrates, developed pulmo-
nary failure, and died within 7 weeks and 3 weeks, respectively,
from AAV detection. AAV was not detected in BAL or tissue
specimens (from lung, gut, liver, kidney, spleen, and lymph no-
des) taken at autopsy from either patient, and the postmortem
examinations showed no cytologic changes suggestive of viral
infection. The lungs in both patients showed evidence of diffuse
Consistent with the finding of budding yeast forms in his BAL
specimens, Candida glabrata was detected by PCR assays of BAL
BAL or lung tissue specimens from patients 3. Of note, patient 1
had PCR evidence of BK virus and adenovirus viremia concom-
itant to AAV detection. Likewise, low-level HHV-6 viremia oc-
curredin close proximityto AAV detection in patients2, 3, and 4.
CMV was not detected in any of these patients.
Other clinical findings were unsubstantial. Patient 4 reported
nausea and diarrhea and was found to have hyperbilirubinemia
and grade 3 graft versus host disease withinone week of his AAV
viremia event and died of unrelated causes on day 297. Patient 2
died on day 1517 of relapsed disease. Neither patient had
abnormal pulmonary complications in association with AAV
AAV can be detected in the blood of HCT recipients, but the
incidence is low, viremia is transient, and AAV does not appear
to play a pathogenic role in organ-specific illness, even in a
Demographic and Virologic Characteristics for 145
Male 84 (58)
Mean age, years 40.7
Myeloablative conditioning145 (100)
Matched related49 (34)
Mismatched related9 (6)
Unrelated 62 (43)
Deaths before day 100 37 (26)
Plasma samples tested, no. 1084
Samples per patient, median (range)8 (1–18)
AAV-positive specimens, no.6
AAV-positive patients, no. (%)4 (2.8)
Duration between transplant and AAV
detection, mean (range), days
Duration of viral detection, median (range), wk 1 (1–3)
Viral load, mean (range), copies/mL,753 (50–2571)
Abbreviation: AAV, adeno-associated virus.
aUnless otherwise indicated, values represent no. (%) of patients.
d JID 2011:204 (1 December)
Table 2. Clinical Characteristics of Adeno-Associated Virus (AAV) Viremia in Hematopoietic Cell Transplant Recipients
PTD of AAV-
Clinical Events in
Week Before or
After AAV Detection
1 48 (M) CML (allo-MUR)64 (1043);
Up to 23 Admitted to the
3 (skin, GI tract,
cocci; C. glabrata
C. glabrata; BK
(360 000) and
viremia (10 032)
on d 64
Died on d 111 of
DAD reported as
cause of death at
2 48 (F)MDSRA
,7 NoneNone2 (skin, GI tract) None Low-level HHV-6
on d 21
Died on d 1517 of
blasts; no autopsy
3 40 (F) AML
,7 Admitted to the
LUL and RLL
None BAL and lung
on d 63
Died on d 95 of
418 (M) ALL (allo-MR,
,7 Reported nausea
and diarrhea as
positive for GVHD
at 9.8 mg/dL with
3 (skin, GI tract,
on d 28
Died on d 297 from
Abbreviations: ALL, acute lymphocytic leukemia; allo, allogeneic; AML, acute myelogenous leukemia; BAL, bronchoalveolar lavage; C. glabrata, Candida glabrata; CML, chronic myelogenous leukemia; DAD, diffuse
alveolar damage; GI, gastrointestinal tract; GVHD, graft versus host disease; HHV-6, human herpesvirus 6; LLL, left lower lobe; LUL, left upper lobe; MDSRA, myelodysplastic syndrome with refractory anemia; MR,
matched related; MUR, matched unrelated; PCR, polymerase chain reaction; P. jiroveci, Pneumocystis jiroveci; PTD, posttransplantation day.
aLimit of detection of the assay is 23 copies/mL .
d JID 2011:204 (1 December)
d BRIEF REPORT
highly Download full-text
important in helping to define the virologic characteristics and
clinical epidemiology of AAV, a virus that is increasingly being
used as a delivery mechanism for gene therapies and newer-
AAV has been an attractive candidate for use as a viral vector,
stemming from its ability to infect diverse human cell lines, the
specificity of its integration into the host genome, and its pre-
sumed nonpathogenicity. However, little work has been done to
evaluate the clinical epidemiology associated with wild-type AAV,
and this virus has not been previously studied in highly immu-
nocompromised patients. Although studies of wild-type AAV
have historically focused on the serologic identification of prior or
latent infection in hospitalized patients [12, 13], we used molec-
ular diagnostic techniques to demonstrate instances of acute AAV
viremia in a cohort of immunocompromised HCT recipients.
Although 2 patients died of pulmonary complications in
proximity to their viremia (both ,7 weeks), AAV was not de-
tected in BAL or lung tissue specimens (or other tissue speci-
mens) from either patient. Further, their autopsy specimens
lacked any histopathologic evidence of viral infection. However,
the number of cases is small, and it is unknown whether AAV
itself would cause histologic changes in human tissue and
whether histopathologic changes, if they occurred, would be
those of the helper virus. Overall, the deaths are plausibly ex-
plained by Aspergillus pneumonia (case 1) and idiopathic
pneumonia syndrome with leukemic relapse (case 3). Given the
history of early Aspergillus infection in patient 3 and the po-
tential for residual disease, this patient may also have died from
fungal pneumonia, although we did not find PCR evidence of
fungi in his lung tissue specimens.
It is interesting that, in addition to having AAV viremia, all 4
patients had evidence of concomitant viremia with HHV-6, BK
virus, or adenovirus, although the viral load was low in 2 of the
patients. The facilitated release in vitro of AAV from its host
genome by proteins from helper herpesviruses or adenoviruses
has been well described , yet there have been very few reports
of in vivo codetection in symptomatic humans. In a small, 40-
person epidemic of adenovirus conjunctivitis and pharyngitis in
1975, AAV was coisolated with adenovirus in secretions from
40% of patients . Our study is the first to document code-
tection of these known helper viruses and AAV in immuno-
compromised patients. More than half of the patients in the
cohort had viremia with other DNA viruses [5, 10].
The strengths of this study are the use of a consecutive un-
selected cohort with weekly prospectively collected plasma
samples, quantitative assessment of AAV in BAL and tissue
potential causes of disease. Limitations include the lack of other
the newly characterized human AAV serotypes from our PCR
analyses . Although our assay covered the most common
immunocompromisedpopulation. This studyis
serotypes used for AAV vectors, it is unknown whether the
frequency or clinical manifestations of the uncovered subtypes
would be any different from those of subtypes 1–8. In summary,
low-level transient AAV viremia can be detected in HCT
recipients, but the incidence is low, and AAV does not appear
to cause any organ-specific illness, even in a highly immuno-
Financial support.This work was supported by the National Institutes
of Health (grants CA18029, CA15704, HL081595, and HL093294).
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
We thank James Ferrenberg and Nancy Wright, for
1. Kotin RM, Siniscalco M, Samulski RJ, et al. Site-specific integration by
adeno-associated virus. Proc Natl Acad Sci U S A 1990; 87:2211–5.
2. Wang XS, Ponnazhagan S, Srivastava A. Rescue and replication signals
of the adeno-associated virus 2 genome. J Mol Biol 1995; 250:573–80.
3. Tobiasch E, Burguete T, Klein-Bauernschmitt P, et al. Discrimination
between different types of human adeno-associated viruses in clinical
samples by PCR. J Virol Methods 1998; 71:17–25.
4. Limaye AP, Huang ML, Leisenring W, et al. Cytomegalovirus (CMV)
DNA load in plasma for the diagnosis of CMV disease before en-
graftment in hematopoietic stem-cell transplant recipients. J Infect Dis
5. Zerr DM, Corey L, Kim HW, et al. Clinical outcomes of human her-
pesvirus 6 reactivation after hematopoietic stem cell transplantation.
Clin Infect Dis 2005; 40:932–40.
6. Musher B, Fredricks D, Leisenring W, et al. Aspergillus galactomannan
enzyme immunoassay and quantitative PCR for diagnosis of invasive
aspergillosis with bronchoalveolar lavage fluid. J Clin Microbiol 2004;
7. Fredricks DN, Relman DA. Paraffin removal from tissue sections for
digestion and PCR analysis. Biotechniques 1999; 26:198–200.
with newly described coronavirus subtypes. Pediatrics 2007; 119:e70–76.
9. Kuypers J, Wright N, Ferrenberg J, et al. Comparison of real-time PCR
assays with fluorescent-antibody assays for diagnosis of respiratory
virus infections in children. J Clin Microbiol 2006; 44:2382–8.
10. Erard V, Storer B, Corey L, et al. BK virus infection in hematopoietic stem
cell transplant recipients: frequency, risk factors, and association with
postengraftment hemorrhagic cystitis. Clin Infect Dis 2004; 39:1861–5.
11. Erard V, Huang ML, Ferrenberg J, et al. Quantitative real-time poly-
merase chain reaction for detection of adenovirus after T cell-replete
hematopoietic cell transplantation: viral load as a marker for invasive
disease. Clin Infect Dis 2007; 45:958–65.
12. Blacklow NR, Hoggan MD, Sereno MS, et al. A seroepidemiologic
study of adenovirus-associated virus infection in infants and children.
Am J Epidemiol 1971; 94:359–66.
13. Erles K, Sebokova P, Schlehofer JR. Update on the prevalence of serum
antibodies (IgG and IgM) to adeno-associated virus (AAV). J Med
Virol 1999; 59:406–11.
14. Schmidt OW, Cooney MK, Foy HM. Adeno-associated virus in ade-
novirus type 3 conjunctivitis. Infect Immun 1975; 11:1362–70.
15. Schmidt M, Voutetakis A, Afione S, et al. Adeno-associated virus type
12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate
proteoglycan-independent transduction activity. J Virol 2008; 82:
d JID 2011:204 (1 December)