By K. Kimberly McCleary
President & CEO, The CFIDS Association of America
In October 2009 CFS was linked to xenotropic murine leukemia virus-related virus, or XMRV, in a paper published by Lombardi et al in Science. A second study published in the Proceedings of the National Academy of Sciences (PNAS) in August 2010 broadened the search, linking CFS to fragments of viruses from the larger family of murine leukemia-like viruses (MLVs). High-profile studies sustained research interest and media attention in this potential association, the role of infectious agents in CFS and immune system abnormalities. Institutions involved with protecting the safety of the blood supply took immediate action to assess the potential risks to blood donors, and investigators concerned about possible viral origins of a wide array of other diseases tested patient blood samples for this family of viruses. Here’s an update on where this research stands today.
To date, more than 20 studies have failed to support the initial observations that associated CFS and the MLV family of retroviruses. The original Lombardi study was partially retracted on Sept. 22, 2011, by Robert H. Silverman, Ph.D. et al, after a rigorous re-examination of some of the samples reported in the 2009 paper determined that they were contaminated with XMRV plasmid, rather than there being naturally occurring virus present. In its Dec. 23, 2011 issue, Science editor-in-chief Bruce Alberts fully retracted the 2009 publication. On Dec. 26, 2011, authors of the 2010 PNAS study retracted their study citing failure (by any group) to replicate the results.
The National Institutes of Health (NIH) has provided funding for two multicenter studies to try to resolve the scientific controversy and assess the safety of the blood supply. The first, known as the Blood XMRV Scientific Research Working Group (SRWG) study, began in November 2009 and was completed in August 2011. Final results were published in Science on Sept. 22, 2011. None of the 19 assays used by nine participating labs was able to distinguish previously XMRV/MLV-positive CFS cases from healthy blood donors or pedigreed negatives on the basis of results for XMRV or the larger family of murine leukemia viruses. Positives detected by just two of the participating labs were found at the same low rates in cases and controls, and results for samples from the same individual were not consistently positive in either laboratory.
The second large NIH-supported study is being coordinated by Ian Lipkin, M.D., at Columbia University. Six clinical sites have provided samples from well-characterized CFS patients and matched healthy controls. Coded samples are being tested under blinded conditions by investigators using their own assay methods and Dr. Lipkin will break the code; any discrepant results will be further evaluated with additional testing. The laboratory investigators involved in the study include Dr. Judy Mikovits (formerly of the Whittemore Peterson Institute), Dr. Francis Ruscetti (National Cancer Institute), Dr. Shyh-Ching Lo (Food and Drug Administration) and William Switzer (Centers for Disease Control & Prevention). The study is due to be completed by mid-2012.
UPDATE (May 22, 2012): Results of testing for XMRV and MLVs by Maureen Hanson, Ph.D.”s group at Cornell University were reported May 21, 2012, in PLoS ONE. She obtained samples from two physicians who specialize in CFS, David S. Bell, M.D. (Lyndonville, N.Y.) and Susan Levine, M.D. (Manhattan, N.Y.), as well as from 12 healthy controls in Ithaca, N.Y. In all, her lab tested 40 CFS patients” samples and samples from 36 healthy controls. Although Dr. Hanson had reported initial positive results in patients” samples more often than controls” at several conferences and meetings, subsequent testing failed to uphold this finding. The PLoS ONE paper concludes, “While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.” The authors suggest that high-throughput DNA sequencing will be more fruitful than PCR for identifying viral pathogens in CFS.
Origins of XMRV
A National Cancer Institute team led by Tobias Paprotka, Ph.D., reported in Science in May 2011 that XMRV likely originated through a laboratory recombination of two mouse viruses in the prostate cancer cell line 22Rv1, between 1993 and 1996. This conclusion is supported by a number of published studies that have used serology to look for evidence of an immune response to XMRV infection, but found none.
The other piece of evidence weighing against XMRV as a human pathogen are multiple published reports of contaminants with mouse DNA in laboratory reagents, supplies and equipment that can show falsely positive results in tests for XMRV and other murine leukemia virus-like viruses (MLVs). A new study published today in PLoS ONE echoes findings from earlier publications about the ability of XMRV to rapidly grow in numerous types of human cell cultures, thus posing a biohazard risk for laboratories that work with XMRV-producing cell lines. The team from several institutions in Barcelona, Spain, led by Marta Curriu, Ph.D., reported that while XMRV replicated in in vitro cultures of able to infect human lymphoid tissue and produce infectious viruses, they were not able to establish a new infection in freshly collected ex vivo tonsil tissues. They found that XMRV replication was controlled by innate antiviral restriction factors in these conditions that closely simulate the natural environment.
Prostate Cancer Studies
Before XMRV was linked to CFS, it was associated with prostate cancer based on a 2006 study by researchers at the Cleveland Clinic and at the University of California-San Francisco. They detected XMRV in tissue from prostate cancer tumors in 8 of 20 subjects with a particular variant of the RNase L cancer susceptibility gene and only 1 of 66 tissue samples obtained from subjects with a different RNase L variant. A dozen follow-on studies of prostate tumor tissues, blood samples from individuals with prostate cancer and prostate secretions yielded mixed results but never attracted the same kind of attention as studies of XMRV in CFS.
On May 16, 2012, the journal PLoS ONE published two studies that help resolve lingering questions about the possible role of XMRV in prostate cancer. The first of these studies is from two members of the Cleveland Clinic group, including Dr. Silverman, that made the original 2006 discovery and collaborators at Abbott and Roswell Park Cancer Institute. They traced the origins of the human prostate cancer cell line, 22Rv1, that was infected with XMRV after multiple passages through nude mice. The tumor tissue used to make that cell line was excised from a patient with prostate cancer in 1992 at Case Western Reserve University known as patient CWR22. The team reporting its results today was able to obtain five paraffin-embedded prostate tumor samples from patient CWR22 for additional testing. Using six testing techniques, the team confirmed that there was no evidence of XMRV in the CWR22 tumor tissues. They conclude, “Our findings conclusively show an absence of XMRV or related viruses in prostate of patient CWR22, thereby strongly supporting a mouse origin of XMRV. While XMRV was originally identified in a study of prostate cancer patients, the sequence of XMRV present in 22Rv1 cells is virtually identical with XMRV cloned using human prostate samples thus suggesting laboratory contamination with XMRV nucleic acid from 22Rv1 cells as the source.”
The second prostate cancer-related PLoS ONE study published on May 16, 2012, was led by Dusty Miller, Ph.D., of the Fred Hutchinson Cancer Research Center. His team reports results of testing samples obtained from the Cleveland Clinic prostate cancer patients, including nine subjects who had previously tested positive for XMRV. They tested blood samples from 29 subjects for infectious XMRV and neutralizing antibodies against XMRV. Prostate secretions from another five subjects were tested for infectious XMRV. They found no evidence for XMRV infection in any sample. They write, “Our results are consistent with the majority of published studies on XMRV, which find that XMRV is not present in humans. The observed low to undetectable XMRV neutralization by human plasma indicates a lack of innate restriction of XMRV replication by soluble factors in human blood.”
Studies of XMRV in Other Diseases
In addition to prostate cancer and CFS, evidence of infection with XMRV has also been looked for in samples obtained from men with HIV, men at risk for HIV infection, individuals with ALS, MS, Bechet’s disease, spondyloarthritis, rheumatoid arthritis, hepatitis C infection, fibromyalgia, lupus, B-cell lymphoma, common types of lymphoid malignancies, transplant patients, fathers of children with autism and children with autism and idiopathic diseases. So far, in these limited studies, no XMRV has been found. A group of researchers in Germany reported finding XMRV-specific sequences in two to three percent of 168 samples from immunocompromised carriers and about 10 percent of samples from 161 immunocompromised patients. In a study of autistic children in Italy in which all of the autism subjects were negative, three of 97 control subjects were positive by PCR for MLV sequences. Neither of these positive reports has been confirmed by other laboratories.
The weight of all evidence points against any threat to the blood supply and one large study conducted by the American Red Cross and reported in Transfusion on Nov. 21, 2011, provides additional evidence that XMRV and MLVs do not pose risks for those in need of blood transfusions. There has been no formal policy change to Food and Drug Administration policy regarding blood donation by individuals with CFS or prostate cancer as a result of the XMRV research; however, the AABB, an organization representing blood banking centers in the U.S. and around the world, has recommended the indefinite deferral of potential blood donors with a past or current history of CFS. This recommendation has been adopted by the American Red Cross and America’s Blood Centers, the two largest blood collectors in the U.S. It is consistent with the CFIDS Association’s long-standing recommendation that individuals with CFS not donate blood, tissues or organs as a precaution to both donor and recipient.
Additionally, a study conducted by researchers at the FDA’s Center for Biologics Evaluation and Research found no evidence of XMRV in a variety of cell lines used for research and vaccine development and production.
The results of the multicenter study testing samples collected from CFS patients and healthy controls being coordinated by Dr. Lipkin (described above) will be published on Sept. 18, 2012 in the open-access online journal of the American Society of Microbiology, mBio. Those results will provide a definitive answer about a causal association of XMRV with CFS. The study has also provided the opportunity to collect, with financial support from the NIH, a bank of samples for future research. As Dr. Lipkin wrote in a Dec. 28, 2011 message about the study, “irrespective of study outcome there will be unprecedented opportunity to explore hypotheses other than [whether the] disease is due to XMRV or MLV infection.”
The CFIDS Association of America is committed to identifying disease-modifying treatment for CFS. The possible link of CFS to XMRV has generated tremendous interest in this serious and life-altering disease and the results of this final multicenter study will determine whether the link merits further investigation. We continue to foster the engagement of scientists interested in viral hypotheses and well-reasoned approaches to improving diagnosis and treatment and, through our Research Institute Without Walls, will turn science into treatment for CFS.
Note: This post was updated on Aug. 27, 2012 to reflect the expected publication date for results of the multicenter study of XMRV/MLVs led by Dr. Ian Lipkin, as announced through his Center for Infection and Immunity”s social media accounts.
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Published studies on XMRV and MLRV findings in human diseases and the general population. AABB. 2 Mar 2012 (date of latest revision). Click this link to access: XMRV Studies Table – March 2012
K. Kimberly McCleary has served as the CFIDS Association”s chief staff executive since 1991.