The Iteration of X

THE ITERATION OF X:
Journal of Virology Study Finds No XMRV

Suzanne D. Vernon, PhD, Scientific Director
K. Kimberly McCleary, President & CEO

The results of the long-awaited study conducted by investigators at the University of Utah, the Fatigue Consultation Clinic and ARUP Laboratories were published May 4, 2011, in the Journal of Virology,¹ the premier journal for virus-related research. Expert and lay summaries of this paper by Shin et al. have begun to populate the Internet. Out first was Vincent Racaniello, PhD, a virology professor at Columbia University who has followed the XMRV story with diligence and balance through his Virology Blog and the popular science podcast “This Week in Virology.”

For the past 19 months, the CFIDS Association has also closely reported the evolving XMRV story and has critically reviewed many of the published papers, identifying the strengths and weaknesses of each one. The Shin study is the most comprehensive and robust effort to detect XMRV and related MLVs in well-characterized CFS patients.

What makes it so?

The investigators scrutinized all previous papers on XMRV and MLV-related viruses, learned from the weaknesses and flaws and designed a study that would find XMRV and related-MLVs if, in fact, these retroviruses were present in human blood. The template for this study was published late last year by the study’s senior author, Ila Singh, M.D., PhD.² That paper was warmly received by the patient community, with many comments about how it formed the basis for a worthy replication effort of the Lombardi³ study published in Science. Dr. Singh had published positive evidence of XMRV in prostate cancer tissue, so her ability to detect the virus in human samples is clear. Although this study is not an exact replica of the methods described in the Science paper, all investigators working on XMRV have modified methods in order to optimize detection of XMRV.

Figure showing the collection, processing and analysis of blood samples from CFS patients and healthy volunteers.

For the Shin study, the investigators recruited 100 CFS patients that met both Fukuda⁴ and Canadian⁵ case definition criteria and they included twice as many (200) healthy controls from the same geographic region. Lucinda Bateman, MD, a highly respected physician with two decades of experience with CFS, evaluated all the patients. Here are some of the reported characteristics of this group of patients:

• Viral-like onset – 72%
• Gender distribution – 68% female; 32% male
• Onset at age 50 or younger – 92% (16% had onset under age 20)
• Average duration of illness – 16 years (range 2-60 years)
• Average duration of illness before consulting Dr. Bateman’s clinic – 9 years
• Average age at time of testing – 34.6 years
• Family history of CFS/fibromyalgia – 17% (females); 13% (males)
• Family history of prostate cancer – 20% for female CFS patients and 14% for male CFS patients (none of the male patients had a history of prostate cancer)
• Scores on Rand SF-36 instrument for symptom severity:
  • Less than 50% physical function subscale – 78%
  • Less than 25% on energy/fatigue subscale – 92%
• Unable to work or attend college, even part-time – 43%
• Receiving full disability – 13%
• Past participation in Ampligen trial – 17%

Blood samples were drawn from all local subjects in the same laboratory, within a three-week period. All samples were blinded, processed and analyzed identically.

The Methods

Samples from 14 CFS patients identified by the Whittemore Peterson Institute (WPI) who had repeatedly tested positive for XMRV at WPI were also included and blood was drawn by an independent phlebotomist at their homes. The paper states, “We report here a repeat testing of samples obtained from CFS patients that were recruited, diagnosed and defined as positive exemplars of XMRV infection by the investigators who performed the original WPI-based study. This testing was performed in an independent laboratory (ours), using many of the same techniques as the original study. To our knowledge, this is the first study to report negative findings after a full repetition of all assay methods in patients who have previously tested positive for XMRV.” Although investigators cite the Science study and state that “we obtained a subset of samples from the original cohort that was used to make the association of XMRV with CFS,” Dr. Judy Mikovits of the WPI told the Wall Street Journal that just two of the 14 patients were the same as those tested in the Science study. The investigators were informed at the completion of this study that these 14 patients had repeatedly tested positive by the WPI.

Multiple sensitive molecular assays were used to test for XMRV and polytropic variants, including quantitative and nested polymerase chain reaction (PCR). They used a DNA plasmid that contained the XMRV genome and amplified multiple regions of the genome to be able to detect variations in viral sequences. They write, “In order to be confident of detecting XMRV in clinical samples, we developed our PCR assays to the robust and reliable standards of clinically used assays.” They also tested for possible contamination by mouse DNA using the sensitive IAP assay developed by John Coffin, PhD, and colleagues.⁶

None of the sensitive quantitative PCR tests they developed gave positive results, so they tested all the samples using the nested PCR methods from the Science and PNAS papers. They used an extra precaution against cross-contamination from PCR products by adding dUTP-UNG to their reactions, a process that has been shown to not affect the reaction itself. The authors state, “Using this assay, we found approximately 5 percent of our samples to be positive for products of the expected size, regardless of whether they were patients or healthy volunteers.” The 5 percent that tested positive changed with each sample set they ran, tipping them off to the fact that positivity was not due to the samples themselves. Sequence analysis identified the positive PCR products to be 95-100% similar to MLV-related viruses. Using the IAP assay, they determined that the samples were not contaminated with mouse DNA. Further testing revealed that the likely source of MLV-related virus sequences was found in two brands of enzymes used in the molecular assays. These enzymes – known as Taq polymerases – are distributed by a variety of manufacturers. The investigators found that two types of Taq from one manufacturer were contaminated with mouse DNA, shown by the IAP assay, just as Furata et al. had reported late last year.⁷ Another manufacturer’s Taq did not contain any IAP sequences. However, the Taq polymerases that tested positive for mouse DNA in this paper were not the same as the one used in the Science paper, which used a Taq polymerase from yet a different manufacturer. The one other published study with positive results for polytropic MLV sequences, by Lo et al.,⁸ reported using a Taq polymerase that in this study was shown to be contaminated with mouse DNA.

Another source of contamination identified in the Shin study was a robotic device used to process samples. This robot had been used months earlier to process XMRV-infected tissue cultures. The contaminated robot was determined to be the source for initial sporadic positive blood samples. Once Taq polymerases were changed and the use of the robot was abandoned in favor of manual processing, no further sporadic positives were detected. The authors note in the discussion section, “Our early false positive findings did have one benefit: they confirmed beyond a doubt that our assay methods were highly sensitive to even tiny quantities of XMRV, and thus we would have every expectation of detecting it if it had been present in any of the samples we tested.”

XMRV particles from Ila Singh, MD PhD

Serological tests were used to look for evidence of an immune response to XMRV in 65 randomly selected samples from 31 patients and 34 healthy controls. They used the culture technique described in the Science paper to attempt to grow the virus from plasma, based on reports from Dr. Mikovits that it was the most sensitive assay for XMRV detection. Dr. Frank Ruscetti (National Cancer Institute) helped adapt the protocol from the original study using the LNCaP cells and a time-intensive, six-week method followed by analysis using Western blot. (Both Drs. Mikovits and Ruscetti are acknowledged in the paper for their assistance with the study.) They handled one culture at a time to prevent cross-contamination of samples. “No XMRV protein or DNA was detected in any of the cultures.”

Their Conclusions

Despite these heroic attempts, XMRV was not to be found by multiple methods and the conclusion reached by these investigators is that “XMRV is not associated with CFS.” They also state that, “prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.”

The positive results from serological assays that indicate an immune system response to XMRV as reported in the Science paper have contributed to doubts about past negative papers. However, many experts, speaking at conferences and writing in the literature, have repeated the observation that Shin et al. make in the Journal of Virology, “It is possible that assays that have found anti-XMRV reactivity in CFS patients are due to cross-reactivity to related antigens.”

The choice of XMRV-positive controls has also been criticized in past studies. A positive control is one that allows investigators to calibrate their methods to get the expected – positive – result. This is an essential control for assays like PCR and its use allows investigators to know that primers are amplifying the correct sequence. For XMRV, an amplified and cloned product called VP62 has been used as the positive control. VP62 was first described by Urisman et al.,⁹ who used PCR to amplify two large DNA fragments that spanned the majority of the XMRV genome derived from a prostate cancer tissue sample; these two overlapping DNA fragments were cloned into two separate plasmids. Shin et al. used an infectious clone called pXMRV1 which was derived by combining the two VP62 plasmids (above) into one, making a single plasmid clone containing the full-length XMRV genome.¹⁰ In this paper, Shin et al. used pXMRV1 to produce XMRV-specific proteins for the ELISA and Western Blot assays and as a positive control for PCR. This clone can be put into cell culture where it produces infectious, wild-type XMRV virus. While PCR products and plasmids are well-known sources of DNA contamination, it is not clear to what degree infectious virus clones that grow to very high levels may contribute to laboratory contamination.

Another perplexing issue has been the higher percentage of positive results in cases compared to controls in the Lombardi et al. and Lo et al. papers. Shin and colleagues suggest the following: “It is possible, as has been suggested before, that patient samples were handled more than control samples and thus had a higher likelihood of contamination. In our study, both patient and control samples were handled in the same manner with the same frequency, in a blinded manner. We also suggest that any planned studies proposed to screen for XMRV carefully check their reagents, equipment, and all possible – and seemingly not possible – sources of contamination with exogenous XMRV and mouse DNA. Obviously, all such studies should be conducted with careful blinding of investigators and staff to prevent unintended experimental bias.”

So, where does this leave the association of XMRV to CFS? Based on an inquiry to staff in charge of two ongoing multi-center studies, it appears that these results will not deter or otherwise affect plans for either the DHHS XMRV Blood Scientific Research Working Group (SRWG) study or the study being coordinated by Ian Lipkin, MD, of Columbia University. Results from the third phase of the SRWG are expected late this summer. The Lipkin study is in the final stages of institutional review and ethical approval, but will take many months once under way. There are also other studies in the pipeline.

There are numerous examples of promising scientific discoveries that do not hold up to repeated replication attempts. This has happened before in CFS and in many other diseases, from breast cancer to multiple sclerosis.¹¹ Especially now, with the widespread use of sensitive molecular techniques, there are likely many lessons to be learned through investigations like these.

Ila Singh, MD, PhD

The Journal of Virology paper and the press release issued by University of Utah about the study end with these important statements:

“…there is still a wealth of prior data to encourage further research into the involvement of other infectious agents in CFS, and these efforts must continue.” (Shin et al.)

“‘These research efforts must continue,’ [Singh] says. ‘Chronic fatigue syndrome is a devastating disease for which a cure needs to be found.’” (Ila Singh as quoted in the University of Utah press release)

The WPI and its collaborators are to be commended for dedicating their expertise and precious resources to an intensified search for infectious causes of CFS. And the search for the cause(s) of CFS must and will continue by talented investigators like those at the University of Utah, WPI and around the world who have become aware and intrigued by mystery and challenge of CFS. Meetings like the NIH-sponsored ME/CFS State of the Knowledge Workshop held last month demonstrate the promising array of studies that are providing important insights into the underlying mechanisms, better ways to define and diagnose CFS and ultimately treatments, cures and prevention.

XMRV has brought unprecedented attention and awareness to the plight of all those who suffer from CFS. This will help ensure that research on this life-altering disease of severe magnitude and impact will accelerate now that its reality is no longer questioned.

References

¹Shin 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 chronic fatigue syndrome. Journal of Virology, 4 May 2011.

²Singh, IR 2010. Detecting retroviral sequences in chronic fatigue syndrome. Viruses. 2:2404-2408.

³Lombardi 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.

⁴Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Annals of Internal Medicine. 1994 Dec 15;121(12):953-9.

⁵Carruthers DM, Jain AK, De Meirleir KL, Peterson DL, Klimas NG, Lerner AM, et al.. 2003. Myalgic encephalomyelitis/chronic fatigue syndrome: Clinical working case definition, diagnostic and treatment protocols. Journal of Chronic Fatigue Syndrome, 11 (1):7-115.

⁶Oakes B, Tai AK, Cingöz O, Henefield MH, Levine S, Coffin JM, Huber BT. Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences. Retrovirology. 2010 Dec 20;7:109.

⁷Sato E, Furuta RA, Miyazawa T. An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR kit is amplified using standard primers for XMRV. Retrovirology. 2010 Dec 20;7:110.

⁸Lo 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. Proceedings of the National Academy of Sciences USA. 2010 Sep 7;107(36):15874-9.

⁹Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL. Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog. 2006 Mar;2(3):e25. Epub 2006 Mar 31.

¹⁰ Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR. XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proceedings of the National Academy of Sciences USA. 2009 Sep 22;106(38):16351-6. Epub 2009 Sep 8.

¹¹Voisset C, Weiss RA, Griffiths DJ. Human RNA “rumor” viruses: the search for novel human retroviruses in chronic disease. Microbiology and Molecular Biology Review. 2008;72:157–196.

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 CFIDS Association’s research program as its scientific director.

K. Kimberly McCleary has served as the Association’s chief staff executive since 1991. 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. McCleary currently serves as a member of two AABB Interorganizational Task Forces on XMRV.

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