The De-Discovery of XMRV

In 2009 the journal Science published a paper titled, “Detection of an infectious retrovirus, xenotropic murine leukemia virus-like virus (XMRV), in blood cells of patients with CFS” by Lombardi et al.[1] Immediately scientific teams around the world tapped into existing stores of CFS patients’ samples to test this reported observation. Blood collectors mobilized to assess whether this pathogen represented a new threat. The first few attempts at detecting XMRV in CFS failed but raised important issues long brushed aside – issues of case definition, the name, sample selection, processing and storage, subset stratification and more.

Ten months later, another remarkable team of investigators published positive evidence in another prestigious journal, the Proceedings of the National Academy of Science. The observation made by Lo et al., was close, but not identical to the original study that launched this area of research, as reported in “Detection of MLV-related virus gene sequences in blood of patients with CFS and healthy blood donors.”[2] Although different, the sequence similarities between XMRV and polytropic murine leukemia virus-related viruses (pMLVs) kept hopes alive that scientists were hot on the trail of the long-elusive cause of CFS.

XMRV-workshop-300x114In the three years that have transpired since the landmark Science publication, more than 30 studies have been conducted on XMRV and CFS and 73 articles have been published in the biomedical literature. There has been one scientific meeting focused exclusively on XMRV. Sessions at annual meetings of several discipline-specific meetings have reported emerging XMRV-related research and a number of policy-related meetings have dealt with issues of blood safety. CFS and XMRV have been frequent headlines in the scientific press and the popular press, including regular reporting from Science’s news staff (by Jon Cohen and Martin Enserink), New York Times (David Tuller), Wall Street Journal (Amy Marcus), Chicago Tribune (Trine Tsouderos) and other outlets.

The abundance of evidence has pointed away from XMRV having a role in CFS and basic research on the virus traced its origins to recombination in a laboratory in the mid-1990s. Several groups have shown that multiple sources of contamination that return falsely positive results for tests of these viruses of mouse origin. Work by dozens of teams across the globe and investments by numerous countries’ governments, academic centers and blood collectors have failed to confirm either the finding of XMRV or pMLVs in CFS and in a wide variety of other disease groups. And late last year, both of the original papers were retracted within five days of one another. However, none of these studies had been designed to definitively answer the question – until today.

A paper titled, “A multicenter blinded analysis indicates no association between CFS/ME and either XMRV or pMLV”[3] was published at 12:01 a.m. today in the American Society of Microbiology’s open access journal, mBio. The title itself makes the investigators’ conclusions evident. The stellar group of authors lends further credence: Harvey Alter, John Coffin and Ian Lipkin are preeminent virus hunters. Judy Mikovits, William Switzer, Francis Ruscetti, Maureen Hanson and Walid Heneine are skilled laboratorians. Nancy Klimas, Anthony Komaroff, Jose Montoya, Lucinda Bateman, Susan Levine and Daniel Peterson are respected physicians who have dedicated their professional careers to caring for CFS patients.

To leave no doubt about the findings, the coordinating academic center, Columbia University’s Center for Infection and Immunity, issued a press release titled, “CFS is not linked to suspect viruses XMRV or pMLV; multi-site blinded study puts to rest the notion that these viruses cause the mysterious ailment.”

This paper represents the ultimate study designed to definitively answer whether a link between XMRV, pMLV and CFS exists. Dr. Lipkin’s team at Columbia has considerable expertise testing viral links to rare and common diseases [4] and he was handpicked by top leadership at the National Institutes of Health to guide the group to a definitive result. The study was planned and executed with great care which explains the extended timetable required to complete it. Clear ground rules were established at the beginning and adhered to throughout.

The six expert clinicians from across the U.S. collected fresh blood samples from 147 individuals with CFS who met both the CDC’s CFS case definition and the Canadian definition for ME/CFS based on a current physical exam and standard lab tests to detect exclusionary conditions. In addition, all the cases enrolled in this study were required to have sudden, viral-like onset to amplify the opportunity to identify XMRV/pMLV. These same clinics were tasked with collecting blood samples from 146 people from within their geographic area to match age, sex and other characteristics of the CFS patients. Earlier studies had left open the possibility of geographic restriction of these agents, so it was important to enroll study subjects from diverse regions of the country.

All the blood samples collected at each site were sent to Columbia University’s Center for Infection and Immunity (CII) where they were processed into components required by the testing laboratories. Most important, they were divided into coded aliquots so that only CII staff could determine which sample came from which individual, whether case or control. Sets of coded samples were shipped to the participating laboratories (CDC, FDA and a team representing the combined efforts of the National Cancer Institute (NCI), Cornell University and Mikovits Consulting) where each group used the assay(s) it had optimized to detect XMRV/pMLV.

Results of tests performed on the coded samples were returned to CII. Study coordinator and renowned virus hunter W. Ian Lipkin, M.D., “broke the code” to match laboratory results with the clinical identity of the samples. His team’s analysis revealed the long-awaited answer: XMRV/pMLV sequences and virus were not detected in a single one of the blood samples from CFS patients or healthy controls using sensitive polymerase chain reaction (PCR) techniques performed by the three lab groups.

There were some positive results returned by the NCI-Cornell-Mikovits team using another method that looked for antibody reactions to these agents. Dr. Francis Ruscetti’s NCI lab tested serum from the coded samples using a flow cytometry-based assay slightly modified from the one he reported in the original 2009 Science paper. The team detected antibodies in human serum but when the code was broken, it revealed equal numbers of CFS cases and healthy controls had these antibodies – nine in each group, or six percent of the subjects. The paper states, “The serology results are more difficult to address given that the assay cannot be validated with plasma from humans with confirmed XMRV or MLV infection. We posit that positive results represent either nonspecific or cross-reactive binding and note that irrespective of explanation, a positive signal does not correlate with case status.”

Nonspecific, cross-reacting antibodies are common and can be the bane of many diagnostic serology platforms. When serology assays for HIV were first developed, serum from healthy, non-HIV infected people sometimes contained antibodies that reacted with HIV proteins in the assay. Blood transfusion studies showed that this non-specific antibody pattern did not correlate with transmission of HIV.[5] It is this type of non-specific reaction that has led to development of HIV screening tests based on antibody testing; positive results from such tests are followed with other testing methods considered to be confirmatory.

To avoid drawing incorrect conclusions about the presence of virus based on the antibody reaction alone, it is imperative to use appropriate control experiments in parallel. In the 2009 study and this multicenter study, a monoclonal antibody produced from a rat (7C10 rat monoclonal antibody) was used in the tests. However, this particular antibody has been shown to cross-react with a number of other different viruses when it was originally produced in the early 1980s.[6] As the authors indicate in the mBio paper, the fact that the positive antibody signal was found in equal numbers of CFS case and healthy control subjects’ serum is strong evidence that it is a sign of nonspecific cross reactivity with no clinical relevance. It is also worth noting that study authors agreed in advance that the only “subjects with two positive results in the same sample type were considered positive for XMRV/pMLV.” The 18 samples that tested positive by this method did not meet the stated standard for a “positive” result as agreed to by all the authors.

The results of the study and the news it brings are certainly not the advances that our community hoped for when the 2009 discovery heralded the possibility of enormous leaps forward in diagnosing and treating CFS. There have been important lessons learned across many of the stakeholder groups over these three years [7], but this is little consolation when answers have been so elusive and so many are suffering.

Scientific discovery is the result of systematic and robust methodology. The most important part of the discovery process is rigorously testing a finding to be certain that it is indisputable. It is actually rare in science that an observation is tested as thoroughly as this one has been, so it gives the authors of this paper – and the community – confidence in the answer they have returned. While this study has indisputably shown that XMRV and pMLVs are not linked to CFS, the XMRV story has demonstrated to the world that more than ever CFS is fertile ground for discovery. We will keep plowing.

References
1. Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science. 2009 Oct 23;326(5952):585-9

2. Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, Alter HJ. Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15874-9.

3. Alter HJ, Mikovits JA, Switzer WM, Ruscetti FW, Lo SC, Klimas NG, Komaroff AL, Montoya JG, Batement L, Levine S, Peterson D, Levin B, Hanson MR, Genfi A, Bhat M, Zheng H, Wang R, Li B, Hung GC, Lee LL, Sameroff S, Heineine W, Coffin J, Hornig M and Lipkin WI. A multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus. mBIO. 2012 Sept/Oct. Vol. 3; issue 5. Link to full text: http://mbio.asm.org/content/3/5/e00266-12

4. Lipkin WI. Microbe hunting. Microbiology and Molecular Biology Reviews. September 2010. Vol. 74; No. 3; 363-377.

5. Genesca J, Shih JW, Jett BW, Hewlett IK, Epstein JS, Alter HJ. What do western blot indeterminate patterns for human immunodeficiency virus mean in EIA-negative blood donors? Lancet. 1989 Oct 28;2(8670):1023-5.

6. Wolff L, Koller R, Ruscetti S. Monoclonal antibody to spleen focus-forming virus-encoded gp52 provides a probe for the amino-terminal region of retroviral envelope proteins that confers dual tropism and xenotropism. J Virol. 1982 Aug;43(2):472-81.

7. Glynn SA, Busch MP, Dodd RY, Katz LM, Stramer SL, Klein HG, Simmons G, Kleinman SH and Shurin SB. Emerging infectious agents and the nation’s blood supply: responding to potential threats in the 21st century. Transfusion. 2012 Jun 13.

Suzanne D. Vernon, PhD, earned her doctorate in virology at the University of Wisconsin at Madison and worked in public health research on infectious diseases for 17 years before joining the Association’s staff in 2007. She has more than 80 peer-reviewed scientific publications on topics including HIV, HPV, cervical cancer and CFS. Dr. Vernon has initiated and participated in numerous international and multidisciplinary research collaborations and she now leads the Solve ME/CFS Initiative’s research program as its scientific director.

K. Kimberly McCleary served as the Association’s chief staff executive from 1991-2013. She has participated in dozens of research and patient conferences on CFS and related conditions. She served on the Department of Health and Human Services CFS Coordinating Committee from 1996 until 2001, has testified before Congress numerous times, and has given scores of media interviews about CFS.

September 18, 2012