Another Turn of the Retrovirus Kaleidoscope
By K. Kimberly McCleary
August 23, 2010
The article by Lo et al. published today in the Proceedings of the National Academy of Sciences (PNAS),1 is perhaps the most anticipated report of research conducted on samples from individuals with chronic fatigue syndrome (CFS) since the condition was formally defined 22 years ago. With colleagues at the Food and Drug Administration (FDA), National Institutes of Health (NIH) and Harvard Medical School, Shyh-Ching Lo and Harvey Alter report finding murine leukemia virus (MLV)-related gag sequences in 32 of 37 (86.5 percent) of CFS patients, compared to 3 of 44 (6.8 percent) healthy blood donors. This study substantiates the report from Lombardi et al.2 published last October in Science that ignited interest in and launched numerous research studies into a possible association between CFS and xenotropic murine leukemia virus-related virus (XMRV). XMRV is a gammaretrovirus of likely mouse (murine) origin that has also been associated with subsets of prostate cancer. It has been described as “the first authentic gammaretrovirus that can infect humans.”3 Since October 2009, four groups have failed to find evidence of XMRV in CFS patients. The authors of the PNAS paper did not find XMRV either; rather, they found evidence of a total of six variants of MLV-related retroviruses in samples collected from CFS patients and healthy blood donors.
In describing the difference between the variants they identified and the highly consistent strains of XMRV found to-date in prostate cancer and CFS, the authors write, “…our analysis revealed three different types of MLV-related sequences in CFS patients. In all three groups, the sequences were more closely related to the sequences of polytropic mouse endogenous retroviruses (mERVs) than to XMRVs...” Polytropic refers to the agent’s ability to infect mice and other species, while the “X” for xenotropic refers to the agent’s ability to only infect non-murine species.
Lo’s team is careful to qualify these results in both the PNAS paper and in a press telebriefing4 hosted by NIH this afternoon. In the paper they write, “It remains to be shown that the association that we have found, using the methods that we have described, can be generalized to a larger group of patients with CFS.” They go on to say, “Even if subsequent studies confirm an association between MLV-like viruses and CFS, that will not establish a causal role for these viruses in the pathogenesis of this illness.” The experiments conducted did not include testing for proof of low-grade infection, and the authors recognize that their own study and the other four investigations of XMRV in CFS have not attempted to fully replicate the Lombardi study in this regard.
The paper concludes with a call for “extensive research investigations of appropriately linked donor-recipient cohorts” to determine whether these agents are blood transmissible and disease-causing in blood recipients. Lo and Alter indicated today that some of these studies are under way in their labs and the laboratories of other groups. We concur with their forceful recommendation for more research. As Lo and Alter indicated today, there remains a great deal of work to be done to understand these agents.
In today’s telebriefing, Lo and Alter also addressed possible reasons for the discrepant results of studies of XMRV and MLVs in CFS. They expressed that chief among the factors to be resolved in studies like these is the syndromic nature of CFS and the subjective nature of the selection of patients for research. The CFS patients’ samples tested in Lo’s lab were obtained in the early 1990s from 42 patients seen in independent CFS-specialty clinics maintained by Anthony Komaroff (Brigham and Women’s Hospital, Boston, Mass.), David Bell (private practice, Lyndonville, N.Y.) and Paul Cheney (private practice, Charlotte, N.C.) for a study of a type of bacteria called Mycoplasma fermentans. As reported in Clinical Infectious Diseases in 1993, the samples were all negative for Mycoplasma fermentans.5 The case definition cited in the 1993 paper is the Holmes definition,6 although the description of cases in the PNAS paper is specifically limited to the 25 patients drawn from Komaroff’s clinic. All 25 of Komaroff’s patients met the Holmes criteria for CFS and 21 of them also met the Fukuda criteria7 published in 1994. Unopened vials from 37 of these original 42 patients were stored by Lo and co-workers for the past 15 years.
After the paper linking CFS and XMRV was published in Science in October 2009, Lo’s team at FDA tested these stored samples for the presence of MLV-related viral sequences including XMRV, using nested polymerase chain reaction (PCR). They recorded positive results in 32 of the 37 (86.5%) samples, although none of the sequences for MLV-related viruses matched those reported by Lombardi et al. in Science. Three of 44 samples obtained between 2003 and 2006 from healthy blood donors living in the Washington, D.C. area also tested positive for MLV-related gag viral sequences; none of these positive results matched the strain of XMRV reported by Lombardi either.
This year, eight of the 25 patients recruited by Komaroff into the 1993 Mycoplasma study were recontacted and fresh blood samples were drawn and tested by Lo. Seven of these eight fresh samples again tested positive for MLV-like viral sequences. This extra step makes Lo et al. the first group to report on MLV-related viral sequences from serially followed individuals including freshly collected blood samples from CFS patients.
The PNAS paper describes the laboratory methods employed by Lo et al. in a comprehensive manner, following protocols by Lombardi et al. in the CFS study of XMRV, and by Urisman et al.8 in the study of XMRV in prostate cancer. They describe the approach taken to minimize the possibility of contamination by mouse DNA in their experiments. Our scientific director, Suzanne D. Vernon, PhD, is preparing a more technical analysis of the methods used and how they compare with other studies. It will be distributed later this week.
Figure from Courgnaud et al.
It is certain that this study will take CFS research in new directions since the results are far from conclusive and present many new questions that warrant investigation. Teams already at work will need to test for virus variants to understand the potential role and mechanisms of a broader group of MLV-related viruses in CFS and other human diseases, as well as asymptomatic healthy controls. This includes other researchers attempting to optimize viral detection assays and those assessing the threat this group of viruses poses to the safety of the blood supply if proven to be transfusion-transmitted. This study’s finding of three positive results from 44 healthy blood donors, or 6.8 percent, is the highest rate of MLV-related viral sequences reported to-date in healthy blood donors. Alter indicated today that they are already at work testing samples from 1,000 healthy blood donors. I serve on the AABB’s Interorganizational XMRV Task Force and can report that this topic is a priority for the professionals responsible for the safety of the blood supply.
An accompanying commentary written by Valerie Courgnaud et al.9 advocates for “proof of principle” clinical trials of a combination of reverse-transcriptase inhibitors to “determine whether safe antiviral regimens can impact the clinical course of CFS and to help determine whether xenotropic or polytropic MLV is causally associated with this debilitating disease.” Discussion about clinical trials of these agents, shown in culture to inhibit XMRV,10 has already begun.11 Randomized controlled trials would help guide patients and physicians to make informed decisions about the woefully inadequate arsenal of helpful therapies available to individuals with CFS while other research advances. On today’s telebriefing, the speakers offered no comment about the timeliness of clinical trials.
This paper has already attracted intense attention from the media, the scientific community and the patient community and more than 130 news articles were posted online four hours after the embargo. Its bumpy path to press is described in an editorial titled, “Patients, patience, and the publication process,” written by PNAS editor-in-chief Randy Schekman.12 We will continue to keep readers informed about the latest developments and news.
Over the days and weeks ahead, dialogue within and across traditional boundaries will deepen our understanding of this important paper and the rapidly growing body of literature. It will shape immediate and longer term priorities for research and will build consensus about these agents, the conditions with which they may be associated and the exact nature of those relationship(s), one to another. But as today's media coverage indicates, few will question whether CFS is real or not. This research has helped lay that controversy to rest, at long last.
1Lo S-C, Pripuzova N, Li B, Komaroff AL, Hung G-C, Wang R, Alter H. (2010) Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proceedings of the National Academy of Sciences. 10.1073/pnas.1006901107
2Lombardi 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 (2009). Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with CFS. Science. Vol. 326, 585.
3Rusmevichientong A and Chow SA. (2010) Biology and pathophysiology of the new human retrovirus XMRV and its association with human disease. Immunology Research. 10.1007/s12026-010-8165-y.
4News advisory: Scientists to discuss research on XMRV in blood, chronic fatigue syndrome. http://www.nih.gov/news/health/aug2010/cc-23.htm
5Komaroff AL, Bell DS, Cheney PR, Lo SC. (1993) Absence of antibody to Mycoplasma fermentans in patients with chronic fatigue syndrome. Clinical Infectious Diseases. 17:1074-1075.
6Holmes GP, Kaplan JE, Nelson MG, Komaroff AL, Schonberger LB, Straus, SE, et al. (1988) Chronic fatigue syndrome: A working case definition. Annals of Internal Medicine. 108, 387-389.
7Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff AL. (1994). The chronic fatigue syndrome: A comprehensive approach to its definition and study. Annals of Internal Medicine, 121 (12):953-959.
8Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL. (2006) Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNaseL variant. PLoS Pathogens, 2:e25.
9Courgnaud V, Battini J-L, Sitbon M, Mason AL. (2010) Mouse retroviruses and chronic fatigue syndrome: Does X (or P) mark the spot? Proceedings of the National Academy of Sciences. 10.1073/pnas.1007944107
10Singh IR, Gorzynski JE, Drobysheva D, Bassit L, Schinazi RF. (2010) Raltegravir is a potent inhibitor of XMRV, a virus implicated in prostate cancer and chronic fatigue syndrome. PLoS ONE. 5:e9948.
11Singh IR. This Week in Virology 94: XMRV with Ila Singh. http://www.twiv.tv/2010/08/08/twiv-94-xmrv-with-dr-ila-singh/
12Schekman R. (2010) Patients, patience, and the publication process. Proceedings of the National Academy of Sciences. 10.1073/pnas.1012027107