XMRV abstracts from the 12th Annual Symposium on Antiviral Drug
Resistance, Nov. 6-9, 2011, Hershey, PA
Presentations-
The Genesis of XMRV through Recombination
http://antiviralresistance.org/abstract25_2011.pdf
Multiple Sources of Contamination in Samples from Patients Reported to
Have XMRV Infection
http://antiviralresistance.org/abstract26_2011.pdf
Poster papers-
POSTER 30- Failure to Confirm XMRV/MLV Nucleic Acid in the Blood of
Patients with Chronic Fatigue Syndrome
http://antiviralresistance.org/abstract_poster30_2011.pdf.
POSTER 31- Large-Scale Screening for Infectious Agents: Finding HCV
and Looking for XMRV
http://antiviralresistance.org/abstract_poster31_2011.pdf
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http://antiviralresistance.org/abstract25_2011.pdf
The Genesis of XMRV through Recombination
Krista A. Delviks-Frankenberry1*, Tobias Paprotka1*, Oya Cing=F6z3,4*,
Sheryl Wildt5, Anthony Martinez6, Hsing-Jien Kung6,7, Clifford G.
Tepper6, Wei-Shau Hu2, John M. Coffin3,4, and Vinay K. Pathak1
1Viral Mutation Section and 2Viral Recombination Section, HIV Drug
Resistance Program, National Cancer Institute at Frederick, Frederick,
MD 21702, USA, 3Department of Molecular Biology and Microbiology,
4Genetics Program, Tufts University School of Medicine, 150 Harrison
Avenue, Boston, MA 02111, USA, 5Harlan Laboratories, Indianapolis, IN
46250, USA, 6Department of Biochemistry and Molecular Medicine and
7Department of Urology, University of California at Davis, Sacramento,
California 95817, USA
The newly discovered retrovirus XMRV (xenotropic murine leukemia
virus-related virus) has been reported to be associated with human
prostate cancer and chronic fatigue syndrome; however, these findings
have not been able to be reproduced. By studying the human prostate
cancer cell line CWR22Rv1, which produces XMRV nearly identical to
those viral sequences identified in patients, and analyzing serial
passages of the original prostate tumor xenograft (CWR22) in nude
mice, we sought to determine where, when and how XMRV arose. We found
that the early passage xenografts did not contain XMRV; however, the
later passage xenografts were XMRV positive, as well as the cell lines
CWR22Rv1 and CWRR1 derived from them. Interestingly, two murine
endogenous proviruses from the host nude mice were identified,
PreXMRV-1 and PreXMRV-2, that shared 99.92% identity with XMRV over
>3.2-kb stretches of their genomes.
We further analyzed the distribution of PreXMRV-1 and PreXMRV-2 in 48
laboratory mouse strains and 46 wild-derived mouse strains: PreXRMV-1
was found mainly in Asian mice, whereas PreXMRV-2 was found mainly in
European mice. None of the 94 mouse strains were XMRV positive. Only
three mouse strains were found to carry both PreXMRV-1 and PreXMRV-2,
two of which were nude and likely used to passage the CWR22 xenograft.
These three mouse strains, as well as the contaminating nude mouse DNA
from the CWR22 xenografts, were found to carry the Xpr1n receptor,
which is nonpermissive to XMRV infection. Thus, together, these
findings show that 1) the original CWR22 prostate tumor did not
contain XMRV, 2) XMRV was generated through a recombination event
between PreXMRV-1 and PreXMRV-2 in which human tumor cells were
required for XMRV to propagate, and 3) the association of XMRV with
human disease is likely due to contamination.
*Authors contributed equally to this work
---------------------------------------------------------
http://antiviralresistance.org/abstract26_2011.pdf
Multiple Sources of Contamination in Samples from Patients Reported to
Have XMRV Infection
M.F. Kearney1, J. Spindler1, A. Wiegand1, W. Shao2, E.M. Anderson1, F.
Maldarelli1, J.W. Mellors3, S. H. Hughes1, S.F.J. Le Grice1, and J.M.
Coffin4
1HIV Drug Resistance Program, National Cancer Institute, Frederick,
MD, 2Advanced Biomedical Computing Center, SAIC, Frederick, MD,
3Department of Medicine, University of Pittsburgh, Pittsburgh, PA,
4Department of Molecular Biology and Microbiology, Tufts University,
Boston, MA
Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was
reported to be associated with prostate cancer by Urisman, et al. in
2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009.
To investigate this association, we independently evaluated plasma
samples from 4 patients with CFS reported by Lombardi, et al. to have
XMRV infection and from 5 healthy controls reported to be XMRV
uninfected. We also analyzed viral sequences obtained from
supernatants of cell cultures reported to contain XMRV after coculture
with clinical samples from 9 patients. A qPCR assay capable of
distinguishing XMRV from endogenous MLVs showed that the viral
sequences detected in the CFS patient plasma matched endogenous MLVs
and not XMRV. Single-genome sequences (N=3D89) from CFS patient plasma
were indistinguishable from endogenous MLVs found in the mouse genome
that are distinct from XMRV. By contrast, XMRV sequences were detected
by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing
of the qPCR product confirmed XMRV not MLV). Single-genome sequences
(N =3D 234) from the 9 culture supernatants reportedly positive for XMRV
were indistinguishable from XMRV sequences obtained from 22Rv1 and
XMRV-contaminated 293T cell-lines. These results indicate that MLV DNA
detected in plasma samples from CFS patients was from contaminating
mouse genomic DNA and that XMRV detected in plasma samples from
healthy controls and in cultures of patient samples was due to
cross-contamination with XMRV (virus or nucleic acid).
--------------------------------------------------------
http://antiviralresistance.org/abstract_poster30_2011.pdf.
POSTER 30
Failure to Confirm XMRV/MLV Nucleic Acid in the Blood of Patients with
Chronic Fatigue Syndrome
E.M. Anderson1, J. Spindler1, A. Wiegand1, F. Maldarellli1, J.W.
Mellors2, G. Simmons3, S.A. Glynn4, M.P. Busch3, M.F. Kearney1, and
J.M. Coffin5 for the Blood XMRV Scientific Research Working Group
1HIV Drug Resistance Program, National Cancer Institute, Frederick,
MD, 2Department of Medicine, University of Pittsburgh, Pittsburgh, PA,
3Blood Systems Research Institute and University of California,
San Francisco, San Francisco, CA, 4Transfusion Medicine and Cellular
Therapeutics Branch, National Heart, Lung, and Blood Institute, NIH,
Bethesda, MD, 5Department of Molecular Biology and Microbiology,
Tufts University, Boston MA
Xenotropic-MLV-related virus (XMRV) and murine leukemia viruses (MLV)
were controversially linked to chronic fatigue syndrome (CFS). To
explore this issue, we independently evaluated coded replicate
samples from 15 subjects previously reported to be XMRV/MLV-positive
and from 15 healthy donors previously determined to be XMRV/MLV
negative. Nucleic acid testing using the XMRV single-copy
assay, similar to the HIV single-copy assay, was performed on plasma
(n=3D68), whole blood (n=3D59), and PBMCs (n=3D27) that were blinded and
provided to us by the Blood XMRV Scientific Research Working
Group. Samples also included 15 spiked positive controls. We detected
XMRV in all of the spiked controls but detected no viral nucleic acid
in any of the coded clinical samples previously reported to be
positive
for XMRV or MLV. These results indicate that previous reports of XMRV
infection are likely the result of laboratory contamination.
---------------------------------------------------------
http://antiviralresistance.org/abstract_poster31_2011.pdf
POSTER 31
Large-Scale Screening for Infectious Agents: Finding HCV and Looking for XM=
RV
Jessica R. Keys1, Peter Leone2, Joseph J. Eron, Jr.2, Myra Brinson3,
Ronald I. Swanstrom4
1Epidemiology Department, Gillings School of Global Public Health,
University of North Carolina, Chapel Hill, NC 27599; 2Infectious
Disease Department, School of Medicine, University of North Carolina,
Chapel
Hill, NC 27599; 3Virology/Serology Unit, North Carolina State
Laboratory of Public Health, Raleigh, NC 27601; 4Microbiology and
Immunology Department; University of North Carolina, Chapel Hill, NC
27599
Background & Objective: Since 2002, North Carolina has employed a
screening program for acute HIV that relies on HIV RNA testing of
pools of HIV-antibody negative samples. We obtained a sample of
HIV-negative serum pools to screen for hepatocellular carcinoma
causative agent hepatitis C virus (HCV), GBV-C, and putative prostate
cancer agent xenotropic murine leukemia virus-like retrovirus (XMRV).
Methods: HIV-negative serum pools of 80 samples (N=3D224) were obtained
from the NC State Laboratory of Public Health and tested for HCV,
GBV-C, and XMRV RNA in parallel using real-time PCR. Because a
fraction of each pool was tested, the detection limit was expected to
range from 15,000-150,000 copies/ml serum.
Results: Of 224 pools, 141 (63%) were HCV positive, 176 (79%) were
GBV-C positive, and 0 were XMRV positive. Viremia was estimated to be
6.4 (IQR: 5.9-7.0) and 6.4 (IQR: 5.7-6.9) log10 copies/ml sera for
HCV and GBV-C, respectively. Assuming the Poisson distribution for the
proportion of negative pools, the estimated prevalence was 1.2% for
HCV and 1.9% for GBV-C in this population. Sequence analysis of
HCV and GBV-C is ongoing.
Conclusions: XMRV was not detected in this HIV at-risk population
representing nearly 18,000 testing adults, lending further support to
growing evidence that XMRV is not circulating among humans. However,
HCV and GBV-C were common in this population, with Poisson-based
estimates in agreement with national prevalence estimates (HCV=3D1.3%
and GBV-C=3D1.7%). These results argue for the use of the HIV testing
program to screen for other viruses of known (HCV) and unknown (GBV-C)
pathogenesis.
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