Neurology Center

A bombshell has just gone off in the continuing debate over XMRV, the virus that may or may not cause chronic fatigue syndrome. Actually, 4 bombshells.

A set of papers out today in Retrovirology (1,2,3,4) claim that many previous studies claiming to have found the virus haven't actually been detecting XMRV at all.

Here's the rub. XMRV is a retrovirus, a class of bugs that includes HIV. Retroviruses are composed of RNA, but they can insert themselves into the genetic material of host cells as DNA. This is how they reproduce: once their DNA is part of the host cell's chromosomes, that cell is ends up making more copies of the virus.

But there are lots of retroviruses out there, and there used to be yet others that are now extinct. So bits of retroviral DNA are scattered throughout the genome of animals. These are called endogenonous retro-viruses (ERVs).

XMRV is extremely similar to certain ERVs found in the DNA of mice. And mice are the most popular laboratory mammals in the world. So you can see the potential problem: laboratories all over the world are full of mice, but mouse DNA might show up as "XMRV" DNA on PCR tests.

Wary virologists take precautions against this by checking specifically for mouse DNA. But most mouse-contamination tests are targeted at mouse mitochondrial DNA (mtDNA). In theory, a test for mouse mtDNA is all you need, because mtDNA is found in all mouse cells. In theory.

Now the four papers (or are they the Four Horsemen?) argue, in a nutshell, that mouse DNA shows up as "XMRV" on most of the popular tests that have been used in the past, that mouse contamination is very common - even some of the test kits are affected! - and that tests for mouse mtDNA are not good enough to detect the problem.

• Hue et al say that "Taqman PCR primers previously described as XMRV-specific can amplify common murine ERV sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection." They go on to show that some human samples previously reported as infected with XMRV, are actually infected with a hybrid of XMRV and a mouse ERV which we know can't infect humans.
• Sato et al report that PCR testing kits from Invitrogen, a leading biotech company, are contaminated with mouse genes including an ERV almost identical to XMRV, and that this shows up as a false positive using commonly used PCR primers "specific to XMRV".
• Oakes et al say that in 112 CFS patients and 36 healthy control, they detected "XMRV" in some samples but all of these samples were likely contaminated with mouse DNA because "all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant IAP long terminal repeat [found only in mice] and most were positive for murine mitochondrial cytochrome oxidase sequences [found only in mice]"
• Robinson et al agree with Oakes et al: they found "XMRV" in some human samples, in this case prostate cancer cells, but they then found that all of the "infected" samples were contaminated with mouse DNA. They recommend that in future, samples should be tested for mouse genes such as the IAP long terminal repeat or cytochrome oxidase, and that researchers should not rely on tests for mouse mtDNA.

They're all open-access so everyone can take a peek. For another overview see this summary published alongside them in Retrovirology.

I lack the technical knowledge to evaluate these claims, no doubt plenty of people will be rushing to do that before long. (Update: The excellent virologyblog has a more technical discussion of these studies.) But there are a couple of things to bear in mind.

Firstly, these papers cast doubt on tests using PCR to detect XMRV DNA. However, they don't have anything to say about studies which have looked for antibodies against XMRV in human blood, at least not directly. There haven't been many of these, but the paper which started the whole story, Lombardi et al (2009), did look for, and found, anti-XMRV immunity, and also used various other methods to support the idea that XMRV is present in humans. So this isn't an "instant knock-out" of the XMRV theory, although it's certainly a serious blow.

Secondly, if the 'mouse theory' is true, it has serious implications for the idea that XMRV causes chronic fatigue syndrome and also for the older idea that it's linked to prostate cancer. But it still leaves a mystery: why were the samples from CFS or prostate cancer patients more likely to be contaminated with mouse DNA than the samples from healthy controls?

Robert A Smith (2010). Contamination of clinical specimens with MLV-encoding nucleic acids: implications for XMRV and other candidate human retroviruses Retrovirology : 10.1186/1742-4690-7-112

Late last year, Science published a bombshell - Lombardi et al's Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. This paper reported the presence of a recently-discovered virus in 67% of the blood samples from 101 people with chronic fatigue syndrome (CFS).

The question of whether people with CFS are suffering from an organic illness, or whether their condition is partially or entirely psychological in nature, is the Israel vs. Palestine of modern medicine - as a brief look at the Wikipedia talk pages will show. So when Lombardi et al linked CFS to xenotropic murine leukaemia virus-related virus (XMRV), they were hailed as heroes by some, less so by others. For some balanced coverage of this paper, see virology blog. Everyone agreed though that Lombardi et al was, as the saying goes, "important if true"...

But it wasn't, at least not everywhere, according to a paper out today in PLoS ONE: Erlwein et al's Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome. The findings are all there in the title - unlike Lombardi et al, these researchers didn't find XMRV in the blood of any of their blood samples from 186 CFS patients.

Still, before people start proclaiming that the original finding has been "debunked", or decrying these results as flawed, some things to bear in mind...

This was a different country. Erlwein et al used patients attending the CFS clinic at King’s College Hospital, London, England. The patients in the original study were drawn from various parts of the USA. So the new results don't mean that the original findings were wrong, merely that they don't apply everywhere. Notably, XMRV has previously been detected in prostrate cancer cells from American patients, but not European ones, so geographic differences seem to be at work. So maybe XMRV does cause CFS, it's just that the virus doesn't exist in Europe, for whatever reason - but bear in mind that even the original study never showed causation, only a correlation. There are many viruses that infect people in certain parts of the world and don't cause illness.

On the other hand, it was a similar group of patients in terms of symptoms: Diagnosing CFS can be difficult, as there are no biological tests to confirm the condition, but Erlwein et al say that

Both studies use the widely accepted 1994 clinical case definition of CFS. Lombardi et al. reported that their cases ‘‘presented with severe disability’’ and we provide quantifiable evidence confirming high levels of disability in our subjects. Our subjects were also typical of those seen in secondary and tertiary care in other centres.

But the first study selected patients with "immunological abnormalities", although we're given few details...

These are patients that have been seen in private medical practices, and their diagnosis of CFS is based upon prolonged disabling fatigue and the presence of cognitive deficits and reproducible immunological abnormalities. These included but were not limited to perturbations of the 2-5A synthetase/RNase L antiviral pathway, low natural killer cell cytotoxicity (as measured by standard diagnostic assays), and elevated cytokines particularly interleukin-6 and interleukin-8.

The biological methods were similar: Both studies used a standard technique called nested PCR. (Lombardi et al also used various other methods, but their headline finding of XMRV in 67% of CFS patients vs just 4% of health people came from nested PCR.) PCR is a way of greatly increasing the amount of a certain sequence of DNA in a sample. If there's even a little bit to start with, you end up with lots. If there's none, you end up with none. It's easy to tell the difference between lots and none.

But there were some differences. The first study only looked at a certain kind of white blood cells, whereas the new study used DNA from whole blood. Also, the first study targeted a larger span of viral DNA - from 419 to 1154:

For identification of gag, 419F and 1154R were used as forward and reverse primers.

Than the second one, which examined the section between positions 411 and 606. As a result, primer sequences used - which determine the DNA detected - were different. However, the authors of the new study claim that they would definitely have detected XMRV DNA if it had been there, because they used the same methods on control samples with the virus added, and got positive results...

The positive control was a dilution of a plasmid with a full-length XMRV (isolate VP62) insert, generously gifted by Dr R. Silverman.

Silverman was one of the authors of the original paper - so hopefully, both research teams were studying the same virus. But (although I'm no virologist) it seems possible that the new study might have been unable to detect XMRV if the DNA sequence of the virus from British patients was differed at certain key ways - the whole point about nested PCR is that it's extremely specific.

Finally, there are stories behind these papers. The first study, that suggested that XMRV causes CFS, was conducted by the Whittemore Peterson Institute, who firmly believe that CFS is an organic disorder and who are now offering XMRV diagnostic tests to CFS patients. By contrast, the authors of the new study include Simon Wessely, a psychiatrist. Wessely is the most famous (or notorious) advocate of the idea that psychological factors are the key to CFS; he believes that it should be treated with psychotherapy.

I'm sure we'll be hearing a lot more about XMRV in the coming months, so stay tuned.

Erlwein, O., Kaye, S., McClure, M., Weber, J., Wills, G., Collier, D., Wessely, S., & Cleare, A. (2010). Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008519

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 chronic fatigue syndrome. Science (New York, N.Y.), 326 (5952), 585-9 PMID: 19815723