From ‘Research 1st', the CFIDS Association research review, 13 September (words by Kimberley McCleary).
Two years ago, a gammaretrovirus called xenotropic murine leukemia virus-like virus (XMRV) was linked to CFS with a high-profile study published in Science by a team of researchers from the Whittemore Peterson Institute (WPI), National Cancer Institute (NCI) and Cleveland Clinic [1]. Today, this study by Vincent Lombardi et al. remains the only one to have shown an association between XMRV and CFS. Research groups in six countries have tested samples from CFS patients and published 15 papers [2-16] that cast doubt on the original data. Through more than 170 publications about XMRV, researchers have documented a better understanding of its origins, life cycle and other properties, yet no links to human disease have been firmly established.
Recent Studies
While several of the early follow-up studies involving samples from CFS patients were small, used broader criteria to define CFS or had other limitations, two major studies published in May 2011 addressed many of the criticisms. The first was from Clifford Shin et al., published May 4 in the Journal of Virology [13]. Konnie Knox et al. published results in Science on May 31 [14]. Both groups tested samples from CFS patients who had tested positive at WPI, yet neither group found evidence of the virus in those samples or others they tested from well-characterized patients using multiple detection methods. Both studies attracted considerable media attention that kept CFS and XMRV in headlines for most of May and early June.
Another group at NCI led by Tobias Paprotka reported in the same issue of Science [17] that XMRV originated through a laboratory recombination of two mouse viruses in the prostate cancer cell line CWR22Rv1. The recombination event occurred between 1993 and 1996. This evidence makes it unlikely that XMRV will be shown to be the sole cause of CFS, which certainly existed before these dates. The XMRV sequences submitted by WPI to GenBank, the public DNA sequence database, are 98-100 percent identical to VP62, an XMRV clone derived from 22Rv1 and used by WPI and the Cleveland Clinic.
The other piece of evidence weighing against XMRV as a human pathogen are multiple 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). Four papers published on Dec. 20, 2010, in Retrovirology [18-21] drew considerable attention to the issue of contamination. The two CFS papers published in May (described above) reported specific sources of contaminants that were encountered in their studies. Information posted to NCI’s website in early June states, “a sample of the XMRV viruses reported in the 2009 article has been cultivated from patient samples and was analyzed at NCI. In contrast to the original findings, the new data suggest it is unlikely that these XMRVs were derived from infected patients. Instead, like the other XMRVs that have been sequenced, they appear to be laboratory contaminants.” Robert Silverman of the Cleveland Clinic told the Chicago Tribune in March 2011 that he was “concerned about lab contamination, despite our best efforts to avoid it.” His lab is conducting additional experiments to test for the possibility. WPI has stated repeatedly that its tests for contaminants have been negative.
By the end of May, doubts about the original report had grown so strong that the editor of Science, Bruce Alberts, requested the authors retract the original paper. They declined to do so, stating it was premature. On May 31, Alberts issued an “Editorial Expression of Concern” [22] that is now tagged to the paper pending the outcome of two large multicenter studies being funded by the National Institutes of Health (NIH) (see below).
Supporting evidence for gammaretrovirus association with CFS has come from two groups who have reported finding sequences consistent with the larger family of gammaretroviruses, but not XMRV specifically. The first team, from the Food and Drug Administration (FDA), NIH and Harvard Medical School, published its findings in August 2010 in the Proceedings of the National Academy of Sciences (Shyh-Ching Lo et al.) [23]. Testing stored samples collected in 1993 for a study looking for mycoplasma, Dr. Lo found 32 of 37 samples from CFS patients positive for MLV sequences. Eight patients from that cohort provided fresh samples and seven of those tested positive again. None of the researchers involved in this study has reported any further data. However, two papers published this summer by groups working independently reported that the sequences obtained from two time points were not consistent with evolutionary changes [24, 25].
The other group, led by Cornell University researcher Maureen Hanson, has presented data at meetings, but its findings have not yet been reported in the literature. The WPI has presented positive data from a study of patients in the U.K., but that has not yet been published, either. News about other unpublished data has circulated through the community and some have charged that positive studies are being unfairly declined by journals, but no specific incidents have been made public.
Clarity Ahead?
To bring greater clarity to the issue, NIH is supporting two studies that involve some of the labs that have published conflicting data.
The first is being coordinated by the National Heart, Lung and Blood Institute and is known as the Blood XMRV Scientific Research Working Group study (SRWG). It is a four-phase study [26] designed to evaluate XMRV detection assays in analytical and clinical samples and to make an initial estimate of XMRV prevalence in blood donors.
Results from Phase I were reported in July 2010 at a meeting of the FDA’s Blood Products Advisory Committee (BPAC). They showed that six participating labs (CDC, Gen-Probe, NCI-Drug Resistance Program, WPI and FDA-Lo and FDA-Hewlett) had comparable sensitivity in their testing methods using coded analytical samples spiked with XMRV.
Phase II results were presented at a BPAC meeting in December 2010. The presentation was repeated days later during a webinar hosted by the CFIDS Association. In Phase II, samples were obtained from four subjects whom WPI indicated were positive for XMRV on multiple occasions and one pedigreed negative control (tested in Phase I). Samples were collected in the same manner and then split into three groups for immediate processing and processing after two days and four days to compare results. Five labs (CDC, Gen-Probe, NCI-Drug Resistance Program, WPI and NCI-Ruscetti) tested the samples under blinded conditions using the assays from Phase I. Results from three labs (CDC, Gen-Probe and NCI-Drug Resistance Program) were all negative. WPI had at least one positive result for three of the four XMRV-positive subjects as well as the negative control. NCI-Ruscetti reported positive results for three of four XMRV-positive subjects, as well as the negative control. The results from WPI and NCI-Ruscetti agreed on two of the XMRV-positive subjects and differed on the other two. One of the aims of Phase II was to determine whether processing time affected testing results; the data indicated that it did not. In spite of the other conflicting data, it was deemed that Phase III would proceed.
This spring, samples were collected from 30 subjects who previously tested positive for either XMRV or MLV sequences, two pedigreed negative controls and 12 blood donor controls. Five analytical controls were included in the panel as well. Eight participating labs (CDC, Gen-Probe, NCI-Drug Resistance Program, WPI, NCI-Ruscetti, FDA-Lo, FDA-Hewlett and Abbott) tested blinded samples according to approved protocols and each has submitted its results to the Blood Systems Research Institute for decoding and analysis. Results will be presented at meetings this fall and submitted to a peer-reviewed journal.
The need for Phase IV (focused on blood donors) will be based on the outcome of Phase III.
The second large study is being sponsored by the National Institute for Allergy and Infectious Diseases. W. Ian Lipkin, M.D., of Columbia’s Center for Infection and Immunity, a renowned pathogen hunter, is coordinating the collection of samples from 100 well-characterized CFS patients and 100 matched controls from four sites around the country. Samples will be processed, blinded and sent to labs at the FDA (Lo), CDC and WPI. Dr. Lipkin will break the code and reconcile the results. Although the study was expected to wrap up before year-end, delays in securing required institutional approvals may push the conclusion of the study into 2012.
Other studies continue as well:
The American Red Cross is collaborating with Gen-Probe and Abbott Laboratories to test samples from 10,000 healthy people in six geographic areas. They will also test samples from 120 recipients of blood donations from more than 4,000 donors. Both donors and recipients will be tested for evidence of XMRV and MLVs. [27]
The NCI has evaluated CFS patients who had been previously tested for XMRV at one of its clinics for a study of the different assays used to detect XMRV.
The FDA’s Center for Biologics Evaluation and Research has conducted a study of the transmission and infection processes of XMRV to address potential safety concerns in cells used to produce vaccines and blood products; results have been submitted for publication.
Based on presentations made earlier this year at the 18th Conference on Retroviruses and Opportunistic Infections (March 2011) and the 15th Conference on Human Retrovirology, HTLV and Related Viruses (June 2011), there are other reports in the publication pipeline.
It remains to be seen whether XMRV will provide the answers to better methods of diagnosis and treatment that were heralded in October 2009. There is no question that the original report of an association has attracted remarkable scientific talent, increased engagement by health agencies and unprecedented awareness of the devastating impact of CFS. No other report among the 5,000+ peer-reviewed articles about CFS has attracted this much attention or such sustained effort to investigate more thoroughly. The debate over XMRV has been polarizing at times, but there is no longer dispute about whether CFS is worthy of scientific endeavor; that, in itself, is progress.
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 (2009). Detection of an infectious retrovirus, XMRV, in blood cells of patients with CFS. Science. Vol. 326(5952), 585-9.
[2] Erlwein O, Kaye S, McClure MO, Weber J, Wills G, Collier D, Wessely S, Cleare A (2010). Failure to detect the novel retrovirus XMRV in CFS. PloS ONE, 5 (1).
[3] Groom HCT, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow JW, Mattes FM, Breuer J, Kerr JR, Stoye JP, Bishop KN (2010). Absence of XMRV in UK patients with CFS. Retrovirology: 10.1186/1742-4690-7-10.
[4] Van Kuppeveld FJM, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJG, Swanink CMA, et al. (2010) Prevalence of XMRV in patients with CFS in the Netherlands: retrospective analysis of samples from an established cohort. British Medical Journal 2010;340:c1018.
[5] Switzer WA, Jia H, Honn O, Zheng HQ, Tang S, Shankar A, Norbert N, Simmons G, Hendry RM, Falkenberg VR, Reeves WC, Heneine W. (2010) Absence of evidence of XMRV infection in persons with CFS and healthy controls in the United States. Retrovirology.
[6] Ping H, Jinmong L, Li Y. (2010) Failure to detect XMRV in Chinese patients with CFS. Virology Journal. 7:224.
[7] Heinrich TJ, Li JZ, Felsenstein D, Kotton CN, Plenge RM, Pereyra F, Marty FM, Lin NH, Grazioso P, Crochiere DM, Eggers D, Kuritzkes DR, Tsibris AMN. (2010) XMRV prevalence in patients with CFS or chronic immunomodulatory conditions. Journal of Infectious Diseases. DOI: 10.1086/657168.
[8] Hohn O, Strohschein K, Brandt AU, Seeher S, Klein S, Kurth R, Paul F, Meisel C, Scheibenbogen C, Bannert N. (2010) No evidence for XMRV in German CFS and MS patients with fatigue despite the ability of the virus to infect human blood cells in vitro. PLoS ONE 5(12): e15632.
[9] Satterfield BC, Garcia RA, Jia H, Tang S, Zheng HQ, Switzer WM. (2011) Serologic and PCR testing of persons with CFS in the United States shows no association with xenotropic or polytropic murine leukemia virus-related viruses. Retrovirology 2011, 8:12doi:10.1186/1742-4690-8-12.
[10] Erlwein O, Robinson MJ, Kaye S, Wills G, Izui S, et al. (2011) Investigation into the presence of and serological response to XMRV in CFS Patients. PLoS ONE 6(3): e17592. doi:10.1371/journal.pone.001759.
[11] Furuta RA, Miyazawa T, Sugiyama T, Kuratsune H, Ikeda Y, Sato E, Misawa N, Nakatomi Y, Sakuma R, Yasui K, Yamaguti K, Hirayama F. (2011) No association of XMRV with prostate cancer or CFS in Japan. Retrovirology. Mar 17;8:20.
[12] Schutzer SE, Rounds MA, Natelson BH, Ecker DJ, Eshoo MW. (2011) Analysis of cerebrospinal fluid from CFS patients for multiple human ubiquitous viruses and XMRV. Annals of Neurology. 69(4): 735-738.
[13] Shin CH, Bateman L, Schlaberg R, Bunker AM, Leonard CJ, Hughen RW, Light AR, Light KC Singh IR. Absence of XMRV and other MLV-related viruses in patients with CFS. Journal of Virology, 4 May 2011.
[14] Knox K, Carrigan D, Simmons G, Teque F, Zhou Y, Hackett J Jr, Qiu X, Luk KC, Schochetman G, Knox A, Kogelnik AM, Levy JA. (2011) No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected. Science. Jul 1;333(6038):94-7. Epub 2011 May 31.
[15] Oakes B, Qui X, Levine S, Hackett J, Huber BT. (2011) Failure to detect XMRV-specific antibodies in the plasma of CFS patients using highly sensitive chemiluminescence immunoassays. Advances in Virology. doi:10.1155/2011/854540
[16] Jerome KR, Diem K, Huang M-L, Selke S, Corey L, Buchwald D. (2011) XMRV in monozygotic twins discordant for CFS. Diagnostic Microbiology and Infectious Disease. doi:10.1016/j.diagmicrobio.2011.06.003
[17] Paprotka T, Delviks-Frankenberry KA, Cingoz O, Martinez A. Kung HJ, Tepper CG, Hu WS, Fivash MJ Jr, Coffin JM, Pathak VK. (2011) Recombinant origin of the retrovirus XMRV. Science. 2011 Jul 1:333(6038);97-101. Epub 2011 May 31.
[18] Hue S, Gray ER, Gall A, Katzourakis A, Tan CP, Houldcroft CJ, McLaren S, Pillay D, Futreal A, Garson JA, Pybus OG, Kellam P, Towers GJ. (2010) Disease-associated XMRV sequences are consistent with laboratory contamination. Retrovirology 2010, 7:111
[19] Sato E, Furata RA, Miyazawa T. (2010) An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR Kit is amplified using standard primers for XMRV. Retrovirology, 7:110
[20] Oakes B, Tai AK, Cingoz O, Henefield MH, Levine S, Coffin JM, Huber BT. (2010) Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences. Retrovirology, 7:109.
[21] Robinson MJ, Erlwein OW, Kaye S, Weber J, Cingoz O, Patel A, Walker MM, Kim W-J, Uiprasertkul M, Coffin JM, McClure MO. (2010) Mouse DNA contamination in human tissue tested for XMRV. Retrovirology, 7:108.
[22] Alberts B. (2011) Editorial expression of concern. Science. 1 July 2011: 35. Published online 31 May 2011 [DOI:10.1126/science.1208542]
[23] Lo S-C, Pripuzova N, Li B, Komaroff AL, Hung G-C, Wang R, Alter H. Detection of MLV-related virus gene sequences in blood of patients with CFS and healthy blood donors. Proceedings of the National Academy of Sciences. 10.1073/pnas.1006901107.
[24] Coffin JM and Cingoz O. (2011) Endogenous Murine Leukemia Viruses: Relationship to XMRV and related sequences detected in human DNA samples. Advances in Virology. 7 Jul 2011.
[25] Katzourakis A, Hue S, Kellam P, Towers GJ. (2011) Phylogenetic analysis of MLV sequences from longitudinally sampled CFS patients suggests PCR contamination rather than viral evolution. Journal of Virology. JVI.00827-11.
[26] Simmons G, Glynn SA, Holmberg JA, Coffin JM, Hewlett IK, Lo S-C, Mikovits JA, Switzer WM, Linnen JM and Busch MP for the Blood XMRV Scientific Research Working Group. (2011) The Blood XMRV Scientific Research Working Group: mission, progress, and plans. Transfusion. 1 Mar 2011. DOI: 10.1111/j.1537-2995.2011.03063.x
[27] Bergoth T, Salminen M, Escriva A-B. Risk assessment on XMRV implications for blood donation. European Centre for Disease Control. 06 July 2011. Page 18.
A chronological listing of XMRV-related articles and publications is available at www.cfids.org/xmrv/resource-listing.asp
Lo and Alter have already made it clear that their paper supports the findings of Lombardi et al., the study headed up by Mikovits.
XMRV was incorrectly named, if people were hoping the name to indicate how similar these human gammaretrovirses are to the types different types of MLVs. It is important that people are aware that the names of these types were not given for scientific reasons. They were subjective and were disproven last year. So there is no scientific reason to believe that xenotropic polytropic and modified polytropic variants are not the same family of viruses. This too is a common grouping for other retroviruses. Take Feline leukaemia viruses (FeLV), the different types are not considered to be different viruses.
Now XMRV is one variant. It is a xenotropic polytropic hybrid. The CDC may have found that it also part ecotropic, research still to be published. In Lombardi Silverman only fully sequenced 3 strains that were all found to be the XMRV variant. The others, from all the other positive patients are still to be fully sequenced. What are they? Lo found polytropic and modified polytropic type sequences. The same as Lombardi did. They also found a third, modified polytropic. This was reported by the WPI/NCI months before as also one of their findings. So they do support each other.
As all the negative papers have failed to replicate proven methods or diagnostically validate their new methods, they remain negative papers. Not papers that can refute the findings of papers that used different methods that were validated. It really is science 101. Any paper that does this cannot seriously be considered as even remotely suggesting issues with Lombardi or Lo et al. It doesn’t matter if you claim to have retested previous positives if your assay has never been shown capable of detecting wild-type virus.
Still, there is no scientific evidence of contamination, but there is evidence that Paprokta et al. failed to report the use of a third assay in the paper that tried and failed to claim that a cell line, not properly investigated was the origin of one variant of XMRV. A cell line that does not contain other strains and variants of human gammaretroviruses. A cell line that has never been in the WPI/NCI/Lo or Alter labs.
The sequence diversity of human gammaretroviruses is also typical of a gammaretrovirus. That is no argument from which to build a case of contamination from. It is also the same for deltaretroviruses and betaretroviruses. All of these rely on colonal expansion to increase their numbers. This makes genetic diversity almost impossible. Many retroviruses in fact have less than 2% diversity. So why is anyone using diversity as argument?