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XMRV-Chronic Fatigue Link Likely Due to Lab Contamination

New studies indicate that the presence of the mouse retrovirus XMRV in blood cell samples from chronic fatigue syndrome patients probably resulted from laboratory contamination.

A study published in Science magazine in October 2009 found an association between a novel mouse retrovirus dubbed xenotropic murine leukemia virus-related virus, or XMRV, and chronic fatigue syndrome (CFS). Vincent Lombardi, Judy Mikovits, and colleagues reported that 67% of CFS patients had evidence of XMRV in their peripheral blood mononuclear cell samples, compared with just 4% of healthy control subjects.

Many people with CFS eagerly embraced Lombardi's research, since finding an infectious cause would offer hope for effective treatment of this debilitating yet still mysterious condition. Later research, for example, demonstrated that some antiretroviral drugs used to treat HIV are also active against XMRV.

Since the original report, however, several studies -- including one published in the online edition of the Journal of Virology last month -- have been unable to replicate Lombardi's results, observing no association between XMRV and CFS.

The debate has continued, but a growing body of evidence weighs in on the side of no significant link, suggesting instead that laboratory error may have led to an erroneous conclusion.

The May 31, 2011, advance online edition of Science features 2 more studies casting doubt on the 2009 findings, prompting the journal's Editor-in-Chief, Bruce Alberts, to issue an "Editorial Expression of Concern" that will be appended to the Lombardi article.

In the wake of these findings, "Individuals with chronic fatigue syndrome need to know that taking antiretroviral therapies will not benefit them, and may do them serious harm," said Konstance Knox, lead author of one of the reports.

Levy Group Finds No Link

In the first study, Knox, pioneering HIV researcher Jay Levy, and colleagues examined blood samples from 61 CFS patients from a single clinical practice (Sierra Internal Medicine in Incline Village, Nevada); 43 of them had previously been identified as XMRV positive in Lombardi's analysis.

Levy's team performed polymerase chain reaction (PCR) and reverse transcription PCR procedures to detect viral nucleic acids, along with assays for detection of infectious virus and virus-specific antibodies.

The researchers found "no evidence" of XMRV or other murine leukemia viruses (MLVs) in the tested blood samples. However, they did observe that these gamma retroviruses were strongly (X-MLV) or partially (XMRV) susceptible to inactivation by blood serum from CFS patients and healthy controls, suggesting that it would be "unlikely" that the viruses could establish infection in humans.

Furthermore -- consistent with previous reports -- they detected MLV sequences in commercial laboratory reagents (chemicals used to facilitate in vitro reactions). These results, Levy and colleagues concluded, "indicate that previous evidence linking XMRV and MLVs to CFS is likely attributable to laboratory contamination."

Virus Recombination

In the second study, Tobias Paprotka and Vinay Pathak from the HIV Drug Resistance Program at the National Cancer Institute and colleagues aimed to learn more about the origins on XMRV, which has also been detected in prostate tumors.

In a laboratory study the researchers examined the human prostate cancer cell lines CWR22Rv1 and CWR-R1, which produce XMRV "virtually identical" to the viruses found in CFS patient samples, as well as a progenitor cell line known as human prostate tumor xenograft (CWR22).

The investigators detected XMRV in the 2 later cell lines and later xenografts (human cells transplanted into mice), but not in early ones. This led them to conclude that XMRV was not present in the original CWR22 prostate tumor cells, but had arisen from recombination of the 2 older related viruses during "passaging" in mice, a procedure in which human cancer cells are injected into mice to grow more tumor tissue for study.

"[O]ur results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event," the study authors wrote.

Expression of Concern

These 2 reports "strongly support the growing view that the association betweenXMRV and CFS described by Lombardi et al. likely reflects contamination of laboratories and research reagents with the virus," Science editor Alberts wrote.

Alberts explained that Lombardi's report "attracted considerable attention, and its publication in Science has had a far-reaching impact on the community of CFS patients and beyond." Because the validity of that study "is now seriously in question," this Expression of Concern will be attached to the original October 23, 2009, report.

"The U.S. National Institutes of Health is sponsoring additional carefully designed studies to ascertain whether the association between XMRV and CFS can be confirmed," he concluded. "Science eagerly awaits the outcome of these further studies and will take appropriate action when their results are known."

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Below is an edited excerpt from a press release issued by the University of California at San Francisco describing Knox and Levy's study and its findings, as well as a history of XMRV and related research.

Mouse Virus Erroneously Linked to Chronic Fatigue Syndrome, UCSF Collaborative Study Finds

San Francisco -- May 31, 2011 -- Two years ago, a widely publicized scientific report plucked an old mouse virus out of obscurity and held it up as a possible cause of chronic fatigue syndrome. According to a new study published today by a group of researchers in California, Wisconsin, and Illinois, that report was wrong.

The mouse virus is not the culprit in chronic fatigue syndrome, said University of California, San Francisco Professor Jay A. Levy, MD, the senior author on the study, published this week by the journal Science.

"There is no evidence of this mouse virus in human blood," said Levy, a professor in the Department of Medicine and director of the Laboratory for Tumor and AIDS Virus Research at UCSF.

Most likely, Levy said, the mouse virus was detected two years ago in blood samples from chronic fatigue syndrome patients because chemical reagents and cell lines used in the laboratory where it was identified were contaminated with the virus.

The bottom line, he said, is that scientists need to keep looking for the real cause -- or causes -- of chronic fatigue syndrome.

This study may have an immediate impact in terms of treatment for some patients with the illness, said Konstance Knox, PhD, of the Wisconsin Virus Research Group in Milwaukee, the first author on the study.

"Individuals with chronic fatigue syndrome need to know that taking antiretroviral therapies will not benefit them, and may do them serious harm," said Knox. "Physicians should not be prescribing antiviral compounds used in the treatment of HIV/AIDS to patients on the basis of a chronic fatigue syndrome diagnosis or a XMRV test result."

A Mysterious Illness

Chronic fatigue syndrome is a somewhat mysterious illness that strikes an estimated 1 million to 4 million Americans with a range of symptoms, including muscle pain, insomnia, memory loss, and overwhelming fatigue. The malaise leaves many with a significantly diminished capacity for physical or mental activity -- often for years, if not decades -- and no amount of bed rest helps.

No definitive cause for the disease has ever been found, and some scientists believe that it is actually the common end stage of a number of different conditions caused by multiple triggers -- trauma, exposure to toxins, or infectious agents, for instance. Several viruses have been named as possible causes, but none ever proved to be the culprit.

In 2009, however, researchers in Nevada and Maryland reported finding traces of a mouse-related virus called xenotropic murine leukemia virus-related virus (XMRV) in about two-thirds of blood samples taken from 101 patients with chronic fatigue syndrome. This virus, they showed, was capable of infecting human cells grown in the laboratory.

Taken together, these results suggested that XMRV could be the cause of chronic fatigue syndrome.

"It gave great encouragement to chronic fatigue syndrome patients," Levy said. "Many believed the cause had finally been found."

The 2009 paper was widely publicized and, according to Levy, it influenced both the direction of federal research spending and the way some doctors began to treat their patients.

Shortly after the paper came out, one of its authors contacted Levy and asked if he could confirm the findings by taking a look at other blood samples taken from patients with chronic fatigue syndrome.

Virus Discovered at UCSF in the 1970s

Levy's experience with the mouse-related virus goes back decades. He and his colleagues were the first to discover xenotropic murine leukemia viruses in the early 1970s. They found that the viruses could infect cells isolated from several animal species, including humans. At the time, their discovery sounded a few alarm bells because health safety officers at the university were concerned about whether these viruses could be transmitted to people. Subsequent tests indicated they could not.

After the initial discovery and studies of this agent over a decade, Levy's research turned to human retroviruses. Once the AIDS epidemic emerged in 1981, much of Levy's work focused on the human retrovirus HIV, which Levy co-discovered in 1983 with two other research groups.

But XMRV came back into focus for Levy in 2009, when he answered a phone call from his former colleague Dan Peterson, MD, of the Whittemore Peterson Institute in Reno, NV, where the 101 samples that yielded traces of the mouse-related virus XMRV had been collected. Peterson, who sees many chronic fatigue syndrome patients in his clinical practice, asked Levy to test blood samples from chronic fatigue syndrome patients.

Levy worked with collaborators at the Wisconsin Viral Research Group in Milwaukee, the Blood Systems Research Institute in San Francisco, the Open Medicine Institute in Mountain View, CA and Abbott in Abbott Park, IL.

They examined blood samples from 61 patients with chronic fatigue syndrome, including 43 who had been previously reported as infected with the mouse-related virus XMRV. Using procedures similar to those performed by the Nevada and Maryland investigators for detecting XMRV and antiviral antibodies, Levy and colleagues found no evidence of XMRV or any other mouse-related virus. Some of the patients were studied on more than one occasion.

They also showed that XMRV was highly unlikely to be an agent of infection. Even though the 2009 paper indicated that it could infect human cells grown in the laboratory, Levy said, the mouse-related virus would not likely cause an infection because human serum quickly kills it.

Finally, Levy and his colleagues hypothesized that the original traces of XMRV found in 2009 were not from the blood samples themselves but from common laboratory reagents or cell lines used in the original experiments. Levy hopes that this finding, also recently shown by others, will help put the controversy to rest.

"With this extensive study, we could not confirm any of the results of the earlier papers," Levy said. He added that even through XMRV is not the cause of chronic fatigue syndrome, evidence remains that some infectious agent could be involved.

"Work should continue to find it," he said.

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Investigator affiliations:

Knox study: Wisconsin Viral Research Group, Milwaukee, WI; Open Medicine Institute, Mountain View, CA; Blood Systems Research Institute, San Francisco, CA; Department of Laboratory Medicine, University of California, San Francisco, CA; Department of Medicine, Hematology/Oncology Division, University of California, San Francisco, CA; Abbott Laboratories, Abbott Park, IL.

Paprotka study: Viral Mutation Section and Viral Recombination Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, MD; Department of Molecular Biology & Microbiology, and Genetics Program, School of Medicine, Tufts University, Boston, MA; Department of Biochemistry & Molecular Medicine, and Department of Urology, University of California at Davis, Sacramento, CA; Data Management Services, Inc., National Cancer Institute, Frederick.

6/3/11

References

K Knox, D Carrigan, G Simmons, J Levy, et al. No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously Identified as XMRV-Infected. Science (abstract  ). May 31, 2011 (Epub ahead of print).  

T Paprotka, KA Delviks-Frankenberry, O Cingöz, VK Pathak, et al. Recombinant Origin of the Retrovirus XMRV. Science (abstract). May 31, 2011 (Epub ahead of print).

B Alberts. Editorial Expression of Concern. Science  . May 31, 2011 (Epub ahead of print).  

Other Sources

University of California San Francisco. Mouse Virus Erroneously Linked to Chronic Fatigue Syndrome, UCSF Collaborative Study Finds. Press release. May 31, 2011.

Science statement. XMRV, Contaminant, not Pathogen? May 31, 2011.