ME Research UK — Energising ME Research

XMRV and ME/CFS — The quest continues

Update at December 2011

The report of a potential retroviral link to ME/CFS, which is estimated to affect 0.2 to 0.4% of the population in developed countries, including between 400,000 and 900,000 people in the USA according to population-based data, certainly caught the world’s attention on 9th October 2009 — no bad thing for an under-researched and often-overlooked illness! The scientific report, entitled “Detection of infectious retrovirus, XMRV, in the blood cells of CFS patients”, appeared online in Science, one of the most prestigious scientific journals in the world, and described the findings of a consortium of researchers from the Whittemore Peterson Institute (WPI, located at the University of Nevada, Reno), the National Cancer Institute (part of the National Institutes of Health) and the Cleveland Clinic, Ohio.

The initial findings

The headline finding of the research paper was that DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), could be detected in the peripheral blood mononuclear cells of 68 out of 101 ME/CFS patients (67%) compared with only 8 out of 218 healthy controls (3.7%). The extent of this difference in proportions is unusual, as it is the norm for scientific researchers to find relatively small yet significant differences between patients and closely matched control groups; in the modern world, novel associations of such magnitude are rarely found between long-standing chronic illnesses and infectious agents. In addition to the headline finding, the researchers determined that XMRV proteins were being expressed in blood cells from ME/CFS patients at very high levels compared with controls, and through cell culture experiments they showed that patient-derived XMRV was infectious and transmissible. So, as well as being the first to show infection with this novel virus in ME/CFS patients, the researchers appeared to have been the first to be able to isolate XMRV particles from the blood, and to show direct transmission of this virus between blood cells — dramatic observations indeed.

What caught the attention of the scientific world is that these observations seem to fit neatly, at least at a first glance, with what is already known about ME/CFS as a chronic illness. For example, viruses related to XMRV have been reported to be involved in damage to blood vessels and nerves, and natural killer cells (historically low in ME/CFS) are said to be susceptible to infection by XMRV. Also, the fact that retroviruses like XMRV are known to be able to activate some other (latent) viruses might explain why ME/CFS has been associated with a range of different viral triggers, such as herpesviruses like Epstein-Barr, over the years. Again, as Dr Judy Mikovits and colleagues pointed out in their paper, some of the most commonly reported features of ME/CFS include neurological symptoms and immune dysfunction with inflammatory cytokine and chemokine upregulation, and some of these observations could be accounted for by infectious XMRV in lymphocytes. The fact that such pieces seemed to fit so well together was suggestive only, however, and a virologist at Tufts University was surely wise to say in New Scientist that while it’s not impossible that infection with this agent might cause a disease with neurological and immunological consequences, there was a long way to go before we knew for sure.

The background

The scientific journey towards this discovery is an extremely interesting one, and includes several strands: prostate cancer, the RNAse L immune pathway, the discovery of the novel virus XMRV, and ME/CFS. XMRV is a human retrovirus similar to HIV, HTLV-1 and a group of endogenous murine leukaemia viruses found in the genomes of wild mice (see the informative presentation on retroviruses by Dr Jones of SAIC-Frederick/NCI-Frederick), and was first identified only in 2006 by Prof. Robert H. Silverman of the Cleveland Clinic, a co-author on the 2009 ME/CFS study. Prof. Silverman initially showed the presence of XMRV in prostate cancer tissue samples (PLoS Pathog, 2006), and subsequent work has confirmed XMRV protein expression in 23% of 334 prostate cancer biopsies (Proc Natl Acad Sci USA, 2009). Importantly, the men with prostate cancer initially studied by Prof. Silverman all had a specific genetic defect in their antiviral defences, the RNase L antiviral pathway which Prof. Silverman had been studying for 30 years, a lifetime’s work of scientific progression described in his fascinating essay, “Journey through the 2-5A/RNase L System”.

RNase L is the terminal enzyme in the 2,5A synthetase/RNase L antiviral pathway, and plays an essential role in the elimination of viral mRNAs. The enzyme has been the focus of research interest in ME/CFS patients for nearly 20 years, and deregulation of this pathway in subsets of ME/CFS patients has been reported extensively in the scientific literature (reviewed by Nijs and Fremont, 2008). In ME/CFS, a wide spectrum of “cleavage” of RNase L can be observed (a phenomenon also seen in multiple sclerosis patients), and such altered RNase L activity profoundly affects cellular physiology, including apoptosis. Overall, an upregulated RNase L pathway in ME/CFS is consistent with an activated immune state and a role for persistent viral infection in the pathogenesis of the disorder — and it is because of these and other findings that many researchers have come to view ME/CFS as primarily a disorder of the innate immune system (see Klimas and Kineru, 2008). It was thanks to the insight of Dr Judy Mikovits and her team at WPI that the potential connection between RNase L dysfunction in XMRV-infected prostrate cancer and in ME/CFS was recognised, and an exploration undertaken to test for the presence of the virus in the banked blood samples in the WPI tissue repository, the largest ME/CFS sample repository in the world.

Subsequent investigations

In the immediate aftermath of their publication, the researchers said that they would continue to refine their test for XMRV. Their plan was to continue their in-depth studies of XMRV to clarify its effects on the human immune system, and to validate clinically a blood test for the detection of XMRV in ME/CFS and other human diseases. And they were to begin the work of determining if any currently approved drugs, such as AZT, might be useful for suppressing XMRV.

At the same time, other independent laboratories across the world began the process of confirming the findings in their own local populations of ME/CFS patients. Since the WPI researchers used samples selected from several regions in the US where “outbreaks of CFS” had been documented (using patients diagnosed on CDC-1994 and 2003 Canadian Clinical criteria ), blood samples from patients in other countries (possibly diagnosed with less stringent criteria) might throw up very different results. For this reason, ME Research UK (with the Irish ME Trust) quickly actioned funding to Swedish researchers to test for the presence of XMRV in Swedish ME/CFS patients (see outline). The results of this investigation were published online in October 2011 (Elfaitouri et al, Plos One, October 2011), followed by an expert review on the subject by Professor Blomberg and colleagues in a special issue of Advances in Virology 2011.

Table. Published studies October 2009 to December 2011
First author, country Journal, date Patients positive for XMRV?
Lombardi, USA Science, October 2009 Yes (67%)
Erlwein, UK PLoS One, January 2010 & March 2011 (re-analysis) No
van Kuppelweld, Netherlands British Medical Journal, February 2010 No
Groom, UK Retrovirology, February 2010 No
Swizer, USA Retrovirology, July 2010 No
Lo, USA Proc Natl Acad Sci, August 2010 No (but 86.5% MLV)
Hong, China Virology Journal, September 2010 No
Henrich, USA J Infect Dis, November 2010 No
Hohn, Germany PloS One, December 2010 No
Satterfield, USA Retrovirology, February 2011 No
Furuta, Japan Retrovirology, March 2011 No
Schutzer, USA Ann Neurol, April 2011 No
Shin, USA Journal of Virology, May 2011 No
Knox, USA Science, July 2011 No
Jerome, USA Diagnostic Microbiology and Infectious Disease, September 2011 No
Ali, USA Virology Journal, September 2011 No
Elfaitouri, Sweden PLoS One, October 2011 No
Fluge, Norway PLoS One, October 2011 No
Cool, Canada Virology, November 2011 No
Simmons, USA Science, November 2011 No
Steffen, Canada PLoS One, November 2011 No

Since the initial report in Science, twenty distinct studies on XMRV in ME/CFS have been published by other researchers in the UK, USA, China, Germany, the Netherlands, Japan, Norway, Sweden and Canada, all keen to test their own populations of patients. As the table above shows, these studies have not been able to find significant levels of the retrovirus in their patient groups, let alone see a dramatic difference in infection between ME/CFS patients and healthy controls. However, one investigation (Lo et al, 2010), instead of finding XMRV itself, detected a more diverse group of closely related murine leukaemia viruses (MLV) in 86.5% of patients compared with 6.8% of controls. Anecdotally, there have been suggestions that XMRV positivity can be found in other populations, but (at December 2011) these data have been presented in poster form only (International Workshop on XMRV at Bethesda, Maryland, September 2010) and have yet to be published.

Of the “negative” studies, two of the most recent are particularly noteworthy. The first (Knox et al) was published in the journal Science in July 2011, and reported finding no evidence of XMRV or MLVs in patients with ME/CFSXMRV (see a responsible summary here). It was published alongside an “Editorial Expressing of Concern” that the validity of the original study (Lombardi 2009), which had also been published in Science, was now “seriously in question”. The second investigation was a $500,000 study, published in Science (Simmons et al 2011), from the Blood XMRV Scientific Research Working Group (BWG) in which none of nine labs could reproducibly detect XMRV or its relatives in the samples. And the same issue of the journal Science which carried the online Simmons et al study contained a “partial retraction” of the original paper, after two coauthors, Silverman and Das Gupta, reported finding contamination in their original samples.

Why have other researchers failed to find XMRV?

As the list of negative published studies has grown, attention has turned to the possible reasons for the inability of independent researchers to confirm the original positive findings, and some of these were first discussed by Dr Robert Silverman, the discoverer of the XMRV virus, in an excellent review (Nature Reviews Urology, July 2010). First, geographical differences in the distribution of XMRV might exist, as is the case with another human retrovirus, HTLV-1, although this explanation becomes increasingly unlikely as studies increase. Second, sequence variations in XMRV, and the existence of divergent or related viruses, are possible and these could easily be missed by many of the methods, in particular polymerase chain reaction. Third, the absence of standardised, highly sensitive methods for the detection of XMRV, coupled with a lack of widely available, positive control human samples might be contributing to the different results obtained between studies; hence the attempts of the NIH’s Blood XMRV Scientific Research Working Group to arrive at an agreed protocol for laboratory testing. However, most debate has centred around the issue of contamination.

In December 2010, four studies appeared in the journal Retrovirology. Collectively, these suggested that XMRV might have originated from the chance recombination of mouse viruses during laboratory experiments, with positive findings reflecting cell-line contamination rather than true infection in humans (see the Wall Street Journal). One of these studies by Hue et al from University College London had compared XMRV gene sequences from the tumour cell line 22Rv1 with sequences found in XMRV-positive patients; virus genetic diversity was found to be greater between cell lines than between patients, suggesting contamination of patient samples by cell line virus in the lab.

Again, in July 2011, a report from Paprotka et al at the National Cancer Institute in Maryland had examined the origins of a previously used human prostate cancer cell line, 22Rv1. The early versions of the cell line, which were still in storage, harboured no XMRV, but DNA matching one half of the virus was found in two different strains of mice that had been studied subsequently. The researchers concluded that XMRV was generated from a unique recombination of pieces of two mouse viruses “that took place around 1993–1996 in a nude mouse”.

The endgame in 2012

Does the list of negative studies above, and the evidence for the contamination hypothesis, mean that XMRV can be ruled out as an important factor in ME/CFS? Not entirely, since there remains the possibility that the levels of markers in blood may be at or below the limit of detection of all assays and/or fluctuate over time as recently described in studies on experimentally infected macaque monkeys (Journal of Virology 2011). And we still await the results of a $2.3 million investigation funded by the US government and led by Prof. Ian Lipkin of Columbia University, in which three labs have each been given 150 patient and 150 control samples for testing in a blinded manner. Only when the blind-codes are cracked early in 2012 will the results be revealed to a scientific world watching with uneasy suspense. Even if XMRV is conclusively found not to be associated with ME/CFS, the search for the real causes must continue apace.

Further information