The team that uncovered the origins of XMRV included (from left) Drs. Krista Delviks-Frankenberry, Tobias Paprotka, and Vinay Pathak, head of NCI's Viral Mutation Section.
Dr. Vinay Pathak was tying up loose ends at his NCI-Frederick lab and packing for a trip abroad last December, when he received an e-mail message that began, "GOT IT."
The message, from Dr. John Coffin of Tufts University, described a genetic analysis of two mouse viruses. Both viruses—one discovered by Dr. Coffin's lab and the other by Dr. Pathak's—resembled parts of the retrovirus XMRV, but they had never been compared.
When the viral DNA sequences were lined up for the first time, Dr. Coffin wrote in the e-mail, they were nearly a "perfect match" with XMRV (xenotropic murine leukemia virus-related virus). "How the DNA sequences fit together was really remarkable," he said recently. "They clicked like two magnets pulling themselves together."
This single experiment tied together months of clues the researchers had been gathering on the origins of XMRV, which was discovered in prostate cancer cells in 2006. Based on their evidence, Drs. Coffin and Pathak now believed that XMRV arose when the two mouse viruses came together (a process known as recombination) during a lab experiment in the 1990s.
In the experiment, a lab worker was growing a patient's prostate tumor cells in mice until the tumor cells could be cultured—the first step in making a colony of cells, or cell line, for research. When one of the samples was eventually harvested from a mouse, the researchers said, the sample contained XMRV. Then, as many labs began to use the cell line, the virus spread.
The details of this detective story were published online in Science on June 2. In the article, the authors concluded that XMRV emerged when the two "parent" viruses recombined in a lab mouse that was being used to grow human tumor samples.
"These results really tell us that XMRV, as we know it, has not been circulating in the human population," said Dr. Hung Fan, a retrovirologist at the University of California, Irvine, who was not involved in the research.
Evidence Points to Contamination
Three years after XMRV was discovered in prostate cancer cells, another group reported finding the virus in patients with chronic fatigue syndrome (CFS). Few labs have been able to confirm this initial report by detecting the virus in patient samples, however, and the possible role of XMRV in human disease has been a source of controversy.
All XMRV isolates reported to date are closely related to the viral sequence found in the prostate cancer cell line known as 22Rv1. If the retrovirus had been replicating in humans, these sequences would contain much more variation, several researchers noted.
Furthermore, the chance of the virus arising independently in another mouse or in the wild is "vanishingly small—about one in a trillion," said Dr. Pathak. This virus is unlikely to exist in nature, so the chance that a person would be exposed to a mouse with the virus is also unlikely, he added.
"Admittedly, we have no direct evidence of a patient sample being contaminated," said Dr. Jonathan Stoye of the United Kingdom's National Institute for Medical Research, who was not involved in the research. "But all of the indirect evidence leads one to believe that contamination is the most likely explanation" for reports of XMRV, he continued.
At a recent workshop on XMRV, co-led by Dr. Stoye, some presenters cautioned that contamination could be a reason that some investigators had detected XMRV in patient samples.
"The [current] study documents a new example of the transfer of viruses from mouse to human cells," said Dr. Stephen Goff, a retrovirologist at Columbia University who also had no role in the study. "This kind of transfer has occurred many times before in other experiments.
"When human tumors are grown in mice," he continued, "the tumors can pick up mouse viruses."
For researchers in the field, a take-home message of the current study is that "anyone who publishes a study saying that XMRV is present in a patient sample needs to look very hard at the data," said the first author, Dr. Tobias Paprotka, a member of Dr. Pathak's lab.
"If it's XMRV, it is almost certainly contamination," added Dr. Pathak.
Testing Prostate Cancer Cells from the Donor
When his investigation began, Dr. Pathak was focused on one question: How had XMRV gotten into the 22Rv1 cell line? As the work progressed, the answer—and the origin of the virus itself—emerged.
Dr. Fan called the study a nice example of "molecular viral archeology," noting that the researchers had tracked down tumor samples in freezers around the country. In California, for instance, Dr. Pathak located a series of tumor samples from the experiment in which the tumor samples had been grown in mice and were later used to create the 22Rv1 cell line.
At Tufts University, Dr. John Coffin and Oya Cingöz, a genetics student in his lab, helped to trace the emergence of XMRV. (Photo by Kelvin Ma)
The researchers discovered XMRV in the later samples, however, indicating that the virus must have emerged during the experiment. Without knowing the risk of contamination, nonvirology labs then handled the prostate cancer cells for a decade.
Once the virus was in the cell line, "it is not hard to imagine how it could have been spread through many labs," said Dr. Coffin, who works part of each month at NCI-Frederick and is a special advisor to the NCI director. In his lab at Tufts, Oya Cingöz, a graduate student in genetics, conducted much of the research.
Although XMRV replicates well in culture and could infect people, it is unlikely that the virus would spread easily from person to person. The reason, Dr. Fan explained in an e-mail, is that humans "have a fairly potent intracellular resistance factor that prevents XMRV infection."
On the Trail of a Mysterious Virus
In the summer of 2009, XMRV was seen as a mysterious and potentially threatening retrovirus. A few months earlier, at the annual Cold Spring Harbor Laboratory meeting on retroviruses, researchers presented a study suggesting that, if the virus were real, millions of people in the United States could be infected.
Because of the prostate cancer link and the possibility that a new retrovirus could be circulating in the population and causing disease, NCI convened a meeting of experts in July 2009.
By September, a plan and funding were in place to develop the tools and resources needed to study XMRV. Dr. Stuart Le Grice of NCI-Frederick had assembled a team of retrovirologists to apply their experience working on HIV to questions about a new retrovirus.
"We looked at XMRV from the perspective that it could be real or it could be contamination," said Dr. Le Grice. "We had no bias going in."
Less than 2 years later, the field has an answer. With the findings published, Dr. Le Grice said, it is important to remember that "there is still no evidence that this virus was ever in patients."
The findings appeared in Science along with a companion report on CFS. In that report, researchers were unable to detect evidence of XMRV in patients whose blood samples had tested positive for the virus in the original 2009 study, also published in Science.
The chance of the virus arising independently in another mouse or in the wild is "vanishingly small—about one in a trillion."
—Dr. Pathak
Since then, at least 10 independent studies have also failed to detect XMRV in patients with CFS.
In an accompanying statement, the editor-in-chief of Science, Dr. Bruce Alberts, "expressed concern" about the 2009 study in Science linking the virus to CFS. The journal, he wrote, awaits the outcomes of two NIH-sponsored studies of XMRV and CFS now under way.
Last week, Dr. Dennis Mangan, who chairs a trans-NIH working group on CFS, said that these studies were expected to continue until completion.
The new reports in Science "are probably not the final word [on XMRV and these diseases], but they are definitive on where we stand at the moment," said Dr. Goff.
It is still possible, he noted, that some individuals may be infected with a virus that contributes to the diseases. Now, however, the burden of proof is on investigators to show that any virus they detect is not the result of contamination and is a contributing factor, he said.
A Cautionary Tale about Contamination
"The XMRV story is a cautionary tale about contamination," said Dr. Pathak. "And as our technology becomes ever more sensitive [for detecting the presence of mouse and viral DNA in patient samples] this will become an even bigger concern."
The XMRV story is also an interesting story of how science happens. Although Drs. Pathak and Coffin were each aware that the other was chasing down a different XMRV-like virus, they never imagined that the viruses would be the two parents of XMRV.
Good fortune played a role as well. As Dr. Le Grice noted, it is conceivable that the two labs might have discovered the same mouse virus rather than both parents, delaying their progress.
On December 17, when Dr. Coffin made the discovery, Dr. Pathak was too busy racing around to even respond to the e-mail until after he had boarded a plane. If Dr. Pathak had received the news a few days earlier, he might have postponed a 2-week vacation to a place without Internet access.
As it happened, he had a wonderful trip. And in the final moments before the flight, he managed to fire off a short reply to Dr. Coffin: "That's awesome!!"
—Edward R. Winstead
Further reading: XMRV and Human Disease Association: Questions and Answers ► XMRV and Human Disease Association: Questions and Answers - National Cancer Institute
FULL-TEXT:
NCI Cancer Bulletin for June 14, 2011 - National Cancer Institute
Biography - Edward Winstead
Edward “Ted” Winstead has written about genes and genomes for more than a decade. He joined the NCI Cancer Bulletin in 2005 after being a reporter for the Genome News Network, where he first wrote about many of the tools now used in cancer research. Ted is a graduate of the Writing about Science program at Johns Hopkins University and a proud member of the NIH Bicycle Commuter Club.
You can follow his tweets from @NCIBulletin and @EdwardWinstead. Selected articles: Biography - Edward Winstead - National Cancer Institute
XMRV and Human Disease Association: Questions and Answers
Key Points
XMRV is a virus that is closely related to viruses known to cause cancer and other diseases in laboratory mice. (Question 1)
The association of XMRV with cancers and diseases in humans has now been proven to be due to contamination from laboratory experiments. (Question 5)
Other viruses might play a role in prostate cancer since it is known that a variety of viruses contribute to the causation of about 15 percent of known human cancers (Question 6).
1. What is XMRV?
XMRV, or xenotropic murine leukemia virus–related virus, is a retrovirus that was first reported in 2006 as a potential cause of prostate cancer. It is closely related to murine leukemia viruses, which cause a wide variety of cancers as well as immunological and neurological diseases in mice. It is known that XMRV is not spread in the air like influenza virus.
2. What is a retrovirus?
A retrovirus is a type of virus that has RNA instead of DNA as its genetic material. In infected cells, the RNA undergoes a process known as reverse transcription, in which DNA copies of the RNA are made and then inserted permanently into the host cells’ chromosomes (organized structures of DNA and protein that are located within a cell’s nucleus). If the viral DNA is expressed inside a host cell, the resulting RNA and protein molecules can be used to generate new virus particles. When retroviruses infect germ cells (sperm or egg), heritable forms of the viruses can arise. Then the integrated viral DNA, or provirus, is passed from parent to offspring. However, there is no evidence suggesting that XMRV can infect human germ cells.
Retroviruses are found in a wide range of mammals, and they can contribute to the development of cancers, such as leukemia or lymphoma, as well as neurological diseases. HTLV-1, the first human retrovirus identified, causes adult T cell leukemia. The second human retrovirus, HTLV-2 has not been associated with any disease. A third, human immunodeficiency virus (HIV), also known as HTLV-3, causes AIDS.
3. Is there a link between XMRV and cancer or other diseases?
XMRV was originally reported in 2006 in tissue samples from men with prostate cancer and was mostly found in combination with a specific defect in a gene called RNASE L, which helps the body defend against viruses.
A 2009 study of more than 300 prostate tissue samples collected from American men reported XMRV in 27 percent of the tumor samples from men who had been diagnosed with prostate cancer and 6 percent of the noncancerous prostate samples in men who had been diagnosed with benign prostatic disease. In contrast, a research group in Germany found no association between XMRV and prostate tumors. The group analyzed 589 prostate tumor samples for the presence of the virus and tested blood samples from 146 prostate cancer patients for antibodies to the virus and found no evidence of the virus or of antibodies to the virus. Another German study, and an Irish study, likewise found no evidence of a link between XMRV and prostate cancer. These differences in findings highlighted the need for more research on this virus.
The virus was subsequently reported to infect people with chronic fatigue syndrome (CFS). A study looking at clusters of CFS cases in Nevada, Florida, and South Carolina reported XMRV in 67 percent of 101 people with CFS, but in less than four percent of 218 healthy people.
4. What steps did NCI and other researchers take to arrive at a better understanding of XMRV’s possible role in disease causation?
2009:
NCI convened an international meeting of scientists in July 2009 to discuss potential public health implications of XMRV. During this meeting, evidence was presented that suggested a link between XMRV and CFS. This evidence pointed to an immediate need for development of rapid and accurate diagnostic tools and methods to identify and analyze XMRV. NCI’s Frederick, Md., laboratories established a XMRV action plan with the responsibility for producing the necessary research tools in both quality and bulk; established reproducible diagnostic platforms; and made their tools, technologies and expertise available to researchers in the broader scientific community, with a goal of undertaking large-scale epidemiological studies.
2010:
NCI-produced tools were made available to outside researchers through the NIH AIDS Reagent Repository (see https://www.aidsreagent.org/Index.cfm). At the same time, independent studies were initiated within NCI to determine whether XMRV was in people who had been diagnosed with CFS or prostate cancer. If a link was found, the goal was to try to establish whether the virus played a role in either condition and, if so, to determine whether antiretroviral therapies that are effective against HIV would display broad-spectrum activity against XMRV as well. NCI was the first to show that in addition to the anti-HIV agent AZT (one of the first drugs used against HIV), tenofovir and raltegravir potently inhibited XMRV replication.
2011:
In contrast to previous findings, and in keeping with reports that subsequently emerged in the literature, two independent NCI studies failed to detect XMRV in humans. In other studies, it was observed that XMRV replication is severely inhibited in human white blood cells, making it doubtful that XMRV replicated efficiently in the blood cells of CFS patients as previously reported. Additional evidence was obtained showing that previous data was potentially confounded by the presence of contaminating mouse DNA, and/or by contamination with XMRV virus in the laboratory.
5. What conclusions can now be made about XMRV and human disease?
Recent studies from NCI and Tufts University laboratories have shown that XMRV originated by recombination between two mouse viruses when human prostate tumors were grown in mice in the mid 1990s as part of experiments to develop an animal model system of human prostate cancer. Because all XMRVs that have been isolated and genetically sequenced are extremely similar to the virus that arose in this original recombination event, they are most likely to be laboratory contaminants derived from this recombinant virus. Using highly-sensitive XMRV DNA detection techniques (similar to those that have become the gold standard for HIV), coupled with methods to detect mouse DNA, NCI researchers tested new, independently-collected serum samples from a small selection of patients reported to be infected in the original 2009 Science publication that hypothesized a connection between XMRV and CFS. This analysis indicated significant levels of mouse DNA contamination in several of these samples; however, there was no evidence that any of the people tested a second time had been infected with XMRV. In addition, 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.
The NCI study is being reported in the same issue of Science as another study of XMRV (Knox et al.) that finds a lack of association between the virus and CFS even in the same patients from a 2009 study.
6. Can a role for viruses in causing prostate cancer or CFS now be ruled out?
While studies now cumulatively and convincingly exclude a role for XMRV in prostate cancer and CFS, they do not rule out the possibility that some other virus or viruses might play a role in either disease. It is known that a variety of viruses contribute to about 15 percent of known human cancers.
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