Sunday, April 17, 2011

Mikovits: Fighting for a Cause

http://www.nature.com/news/2011/110314/full/471282a.html
http://www.nature.com/news/2011/110314/pdf/471282a.pdf

Published online 14 March 2011 | Nature 471, 282-285 (2011) |
doi:10.1038/471282a
News Feature
Virology: Fighting for a cause

When Judy Mikovits found links between chronic fatigue syndrome and a virus, the world took notice. Now, she's caught between the patients who believe her work and the researchers who don't.


On a sunny January afternoon in Santa Rosa, California, a small crowd
waits patiently for Judy Mikovits to arrive. She is scheduled to
deliver a talk on a mysterious virus called XMRV, which she believes
underlies chronic fatigue syndrome. Although she's two hours late —
held up by fog at San Francisco International Airport — not a single
person has left. And when she arrives, they burst into applause.

To a rapt audience, she gives a chaotic and wide-ranging talk that
explores viral sequences, cell-culture techniques and some of the
criticisms that have been thrown at her since she published evidence1
of a link between XMRV and chronic fatigue in 2009. Afterwards,
Mikovits is swarmed by attendees. A middle-aged woman who spent most
of the talk in a motorized scooter stands up to snap pictures of her
with a digital camera. Ann Cavanagh, who has chronic fatigue and has
tested positive for XMRV, says that she came in part for information
and in part to show her support for Mikovits. "I just wish there were
a hundred of her," Cavanagh says.

The event was "surreal", says Mikovits, a viral immunologist at the
Whittemore Peterson Institute for Neuro-Immune Disease (WPI) in Reno,
Nevada. She is discomfited by the attention from patients, which at
times borders on adulation. But her reception among scientists has
been markedly cooler. Numerous follow-up studies have found no link
between the virus and the disease; no group has published a
replication of her findings; and some scientists argue that XMRV is an
artefact of laboratory contamination. Now, even some of Mikovits's
former collaborators are having second thoughts.

Mikovits has dug in, however, attacking her critics' methods and
motives. She says that their distrust of her science stems from doubts
about the legitimacy of chronic fatigue syndrome itself. Chronic
fatigue, also known as myalgic encephalomyelitis, affects an estimated
17 million people worldwide, but it is extremely difficult to
diagnose. Many with the disorder are told that their symptoms — which
include exhaustion, joint and muscle pain, cognitive issues, and heart
and respiratory problems — are psychosomatic. "I had no idea there was
that much bias against this disease," Mikovits says.

The stakes are high and many are taking the risks seriously. Several
countries have barred people with chronic fatigue from donating blood
in case the virus spreads (see 'Something in the blood'). And the US
government has launched a US$1.3-million study to investigate the
link. Patients are already being tested for XMRV, and some are taking
antiviral drugs on the assumption that the virus causes chronic
fatigue by attacking their immune defences. Many say that such action
is premature, but Mikovits is steadfast. "We're not changing our
course," she says.

First findings

In October 2007, Mikovits attended a prostate-cancer meeting near Lake
Tahoe, Nevada, where she met Robert Silverman, a virologist at the
Cleveland Clinic in Ohio. Silverman co-discovered XMRV, which stands
for xenotropic murine leukaemia virus-related virus2. While examining
human prostate tumours, he and his collaborators found genetic
sequences that resemble retroviruses found in the mouse genome. Like
all retroviruses, XMRV rewrites its RNA genome into DNA on infection,
then slips the DNA into the genomes of host cells. Ancient remnants of
such viruses litter animal genomes. But the only active retroviruses
conclusively linked to human disease are HTLV-1, which causes
leukaemia, and HIV.

At the meeting, Silverman was presenting research linking XMRV to
deficiencies in a virus-defence pathway. Mikovits recalled that the
same pathway was weakened in some patients with chronic fatigue. She
wondered whether the prostate-tumour virus could also be behind
chronic fatigue. After the meeting, Silverman sent Mikovits reagents
to test for XMRV.

The idea excited Mikovits, but she had other priorities. After stints
in industry and at the US National Cancer Institute (NCI) in Maryland,
she had recently joined the WPI to lead its research programme. The
WPI was founded in 2006 by physician Daniel Peterson, an expert on
chronic fatigue, and by Annette Whittemore, the wife of a
well-connected Nevada businessman, whose daughter Andrea has had
chronic fatigue for more than 20 years. The Whittemores spent $5
million establishing the WPI, and several million more to support
Mikovits's research, which has attracted few other grants.

At the WPI, Mikovits established a sample collection from Peterson's
patients and began screening it for signs of an infection. A litany of
pathogens has been linked to chronic fatigue over the years, including
Epstein-Barr virus, Borna disease virus, human herpes virus 6 and
HTLV-2. None panned out. Still, the disorder bears some hallmarks of
an infection. Many patients report acute illness before chronic
symptoms appear, and their bodies often show signs of an immune system
at war. The disease can also crop up in apparent outbreaks, including
one characterized by Peterson near Lake Tahoe in the 1980s.

Just before Christmas 2008, Mikovits turned her attention to
Silverman's reagents. She and her postdoc, Vincent Lombardi, known as
Vinny, asked a graduate student to test for XMRV DNA in white blood
cells from some of the most seriously ill people being studied at the
WPI.

The first try turned up just two positives out of 20. But by tweaking
the conditions of the test, Mikovits says her team found XMRV in all
20. "Vinny and I looked at each other and said, 'Well, that's
interesting'," she says. They spent the next few weeks convincing
themselves that they were onto something, and soon conscripted
Silverman and Mikovits's former mentor at the NCI, Frank Ruscetti, to
help prove that XMRV infection was behind chronic fatigue.

"We really retooled our entire programme and did nothing but focus on
that," she says. They kept the effort under wraps, dubbing it 'Project
X'. Even Peterson and the Whittemores weren't clued in. Mikovits says
that the secrecy was necessary because her team also found XMRV in the
blood of some healthy people, raising concerns about blood products.
She hoped to build an airtight case because she worried that sceptical
public-health officials would undermine her work.

In May 2009, the team submitted a paper to Science reporting the
identification of XMRV genetic material in two-thirds of the 101
patients with chronic fatigue they had tested and in 3.7% of 218
healthy people. They also included data suggesting that infected white
blood cells could pass the virus on to uninfected cells.

Reviewers wanted more evidence: a clear electron micrograph of
virus-infected cells, proof that patients mounted an immune response
to the virus, an evolutionary tree showing XMRV's relationship to
other viruses and the locations where viral DNA was integrating into
patient genomes. Mikovits's team went to work. "None of us took any
time off, not even a weekend," she says. They resubmitted the paper in
early July with everything the reviewers had asked for, except the DNA
integration sites, which many scientists consider a gold standard in
proving a retroviral infection.

Later that month, NCI officials who had learned about the work invited
Mikovits to give a talk at a closed-door meeting with other XMRV
researchers and government scientists. "When I finished speaking you
could've heard a pin drop," she says. Mikovits says she thinks at
least one of her manuscript's reviewers was at the meeting, because
soon after, she got a call from a Science editor. Their paper had been
accepted.

Jonathan Stoye, a retrovirologist at the MRC National Institute for
Medical Research in London, wrote a commentary about the paper for
Science3. He had never heard of Mikovits, but Frank Ruscetti's name on
the paper gave him confidence, he says, and "if it were true, it was
clearly very important". Stoye's co-author John Coffin, a
retrovirologist at Tufts University in Boston, Massachusetts, says he
was satisfied with the data and thought it was time to "let the field
and public chew on them".

The BBC, US National Public Radio, The New York Times, The Wall Street
Journal and dozens of other news outlets covered the research.
"Prostate cancer pathogen may be behind the disease once dubbed
'yuppie flu'," Nature announced on its news website the day the paper
came out. Phoenix Rising, a forum for patients with chronic fatigue
that has become a hub for all things XMRV, called the work a "game
changer", and patients flocked to learn more about a virus that they
hoped would explain their condition. But others, including Britain's
leading chronic fatigue patient group, urged caution until more
research buttressed the link.

The first negative findings started to arrive in January 2010 —
failing to find XMRV in 186 people with chronic fatigue from the
United Kingdom4. A month later, a team including Stoye published a
paper5 showing no evidence of XMRV in more than 500 blood samples from
patients with chronic fatigue and healthy people. One day later, the
British Medical Journal accepted a paper reporting more negative
results in Dutch patients6. Studies began piling up so fast that
Coffin made a scorecard to show at talks. "I've lost count now," he
says.

Mikovits says that the discrepancies can be explained by differences
in the geographical distribution of XMRV or in the methods used.

The most common way to detect XMRV is PCR, or polymerase chain
reaction, which amplifies viral DNA sequences to a level at which they
can be identified. Mikovits and her team used this method to detect
XMRV in some of their patients, but she contends that the most
sensitive way to detect the virus is to culture patients' blood cells
with a cell line in which the virus replicates more quickly. This
should create more copies of the virus, making it easier to detect
with PCR and other techniques. She says that none of the negative
studies applied this method exactly, a fact that annoys her. "Nobody's
tried to rep-li-cate it," she says, sounding out each syllable for
emphasis.

In summer 2010, some evidence emerged in Mikovits's corner. Harvey
Alter, a hepatitis expert at the NIH's Clinical Center, and his team
identified viruses similar to XMRV in 32 of 37 people with chronic
fatigue and in 3 of 44 healthy people. They were preparing to publish
their results in the Proceedings of the National Academy of Sciences.
But scientists at the Centers for Disease Control and Prevention (CDC)
in Atlanta, Georgia, were about to publish a negative report. The
authors delayed publication of both papers7,8 for several weeks to
assess discrepancies. The move agitated Mikovits as well as the
chronic-fatigue community, who suspected that important data were
being suppressed.

When Alter's work came out in late August7, Mikovits was ecstatic, and
the WPI released a YouTube video of her touting it. For other
researchers, however, the new paper had shortcomings. The viral
sequences from Alter's paper differed from XMRV, says Greg Towers, a
retrovirologist at University College London. "He doesn't get
variation, he gets a totally different virus." Towers says that mouse
DNA, which is chock-full of virus sequences like those Alter's team
found, probably contaminated their samples, which were collected in
the 1990s. But Alter says that his team found no contamination from
mouse DNA and recovered the same viral sequences from the same
patients sampled a decade later.

Contamination became a dirty word for Mikovits. Just before Christmas
2010, Retrovirology published four papers9,10,11,12 that highlighted
laboratory contamination as a possible explanation for her findings.
One showed, for example, that mouse DNA contaminates an enzyme from a
commercial kit commonly used for PCR. Coffin, an author on two of the
Retrovirology papers, urges caution against over-extrapolating. These
papers do not say that contamination explains Mikovits's results, he
says, just that extreme care is required to avoid it.

Towers and his colleague Paul Kellam, a virologist at the Wellcome
Trust Sanger Institute near Cambridge, UK, are less charitable,
however. Their study12 showed that the XMRV sequences that Mikovits
and Silverman had extracted from patients lacked the diversity
expected of a retrovirus that accumulates mutations as it passes
between patients. "This doesn't look like an onwardly transmittable
infectious virus," says Kellam. A press release for the paper issued
by the Sanger Institute put it more bluntly: "Chronic fatigue syndrome
is not caused by XMRV."

Mikovits is riled when the topic turns to Towers's paper over dinner
one night in Reno — "Christmas garbage", she calls it. Contamination
cannot explain why her team can reproduce its results both in her lab
in Reno and at Ruscetti's at the NCI, she says. Her team checks for
contamination in reagents and in the cells it grows the patients'
samples with. She says that her team has also collected viral
sequences that will address Towers's and Kellam's criticism but that
it hasn't yet been able to publish them. Meanwhile, an unpublished
study of patients in Britain with chronic fatigue bears out the link
to XMRV, she says. "I haven't for one second seen a piece of data that
convinced me they're not infected."

Jay Levy, a virologist at the Univer­sity of California, San
Francisco, has a window in his closet-sized office that looks out into
the laboratory where, in the 1980s, he became one of the first
scientists to isolate HIV. After his discovery was scooped by other
researchers, Levy turned his attention to chronic fatigue and started
a long but fruitless search for an infectious cause.

Now, Levy is putting the finishing touches on what could be the most
thorough response yet to Mikovits's Science paper, adopting the same
cell-culture techniques to detect the virus and using samples from the
same patients. He's done this with the help of Daniel Peterson, who
left the WPI in 2010 for what Peterson says are "personal reasons".
Peterson has questioned the institute's singular pursuit of XMRV, a
research direction that was pursued without his consultation.

Mikovits says that she kept the XMRV work secret from Peterson over
fears he would tell his patients, and left his name off the original
Science manuscript until a reviewer questioned the omission. When
asked whether that episode contributed to his departure, he says, "I
was surprised at the secrecy and lack of collaboration." As for his
motivation to team up with Levy: "I'm just trying to get to the truth.
It's my only motive, because this is such a deserving group of
patients who need to know what's going on."

Others, too, are rallying for a definitive answer. Ian Lipkin, a
microbial epidemiologist at Columbia University in New York, has a
reputation for getting to the bottom of mysterious disease–pathogen
links. His team debunked the association between Borna disease virus
and chronic fatigue, for example. Now he is spearheading the
$1.3-million effort funded by the US government. He is leaving the
testing to three labs: Mikovits's at the WPI, Alter's at the NIH and
the CDC. Each will receive coded samples of white blood cells and
plasma from 150 patients with chronic fatigue and from 150 healthy
controls. The labs will test for XMRV using their method of choice.
Lipkin will crunch the data and unblind the samples.

But even if a study confirms the link to chronic fatigue, it won't be
able to determine whether the virus is the cause. XMRV could, for
example, be an opportunistic infection affecting those whose immune
systems are already dampened by chronic fatigue. Even Mikovits can
only hypothesize as to how it might cause disease.

The virus might not even exist as a natural infection. At a retrovirus
conference this month in Boston, Massachusetts, Coffin and his
colleague Vinay Pathak at the NCI in Frederick, Maryland, presented
data showing that XMRV emerged in the 1990s, during the development of
a prostate-tumour cell line called 22Rv1. Developing the line involved
implanting a prostate-tumour sample into mice, retrieving cells that
might divide indefinitely and repeating the process. But looking back
at DNA samples taken throughout the cell-line's development showed
that human cells became infected only after passing through several
different mice. Importantly, XMRV's sequence seems to have come from
two different mouse strains. "They just sort of snapped together like
two puzzle pieces," says Coffin, an event extremely unlikely to have
happened twice.

XMRV sequences retrieved from patients with prostate cancer and
chronic fatigue — including some who have had chronic fatigue since
the mid-1980s — are nearly identical to the virus from 22Rv1 cells.
The implication, says Coffin, is that this virus, born in a
laboratory, has probably been infecting samples for more than a
decade, but not people. "Although people on the blogs aren't going to
believe me, I'm afraid this is by far the most reasonable explanation
for how XMRV came to be," says Coffin, who hoped that the association
with chronic fatigue would pan out and still thinks some pathogen
other than XMRV could explain the disease.

Silverman, who no longer works with Mikovits, says that he wasn't
using 22Rv1 cells when XMRV was discovered. Nonetheless, the work has
rattled his confidence in XMRV's link to both prostate cancer and
chronic fatigue.

Mikovits, however, is undeterred. The WPI owns a company that charges
patients up to $549 to be tested for XMRV, and Mikovits believes that
patients who test positive should consult their doctors about getting
antiretroviral drugs normally prescribed to those with HIV. Levy and
others worry that she is overreaching. "That's scary for me. These
antiretroviral drugs are not just like taking an aspirin," he says.
Mikovits argues that they might be some patients' only hope. "The
people who we know they're infected should have a right to get
therapy," she says, "They have nothing. They have no other choice."

Context and debate

Back in her Reno laboratory two days after the talk in Santa Rosa,
Mikovits examines a stack of small plastic flasks under a microscope.
Some contain patient cells that she hopes will turn into cell lines
and churn out XMRV. "On Wednesdays I get to take care of my cells, and
that's where I'm the happiest," she says.

She has just come off the phone from a sobbing patient infected with
XMRV whose symptoms had worsened. "They call me every single day,"
Mikovits says. "I don't do science any more. I spend so much time
trying to understand the patients, to understand this disease. People
have moved to Reno to be here," she says. They've left gifts: stuffed
animals, and stacks of bumper stickers that say "Today's Discoveries,
Tomorrow's Cures" and, more boldly, "It's the virus XMRV".

Mikovits clearly shares in the frustration of those with chronic
fatigue who have been marginalized over the years and told that their
disease is not real. She says that this disbelief in the disorder
drives the criticism of her work. Kellam and the others say that this
isn't true. They don't deny the existence of the syndrome or even the
possibility of an infectious origin. "What we're trying to understand
is the aetiology," Kellam says. "It's a scientific debate."

Mikovits says that she's analysed all the papers critical of her work
and found flaws in each of them. Nevertheless, she's quick to endorse
findings that support her work. She claims that Coffin and Pathak's
study, for example, "says nothing about human infection". Yet new work
presented at a different meeting that found XMRV using next-generation
DNA sequencing offers "no doubt it's not contamination — that the
whole story's real", she says.

Despite the growing choir of sceptics, Mikovits says that she has
simply seen too many data implicating XMRV and other related viruses
in chronic fatigue to change her mind. For her supporters, that
steadfastness offers legitimacy and hope. "The scientists are moving
forward," she announced at her talk in Santa Rosa, "and I think the
politics will go away shortly."
The crowd responded with vigorous
applause.

Ewen Callaway writes for Nature from London.

References
1.Lombardi, V. C. et al. Science 326, 585-589 (2009).
2.Urisman, A. et al. PLoS Pathog. 2, e25 (2006).
3.Coffin, J. M. & Stoye, J. P. Science 326, 530-531 (2009).
4.Erlwein, O. et al. PLoS ONE 5, e8519 (2010).
5.Groom, H. C. et al. Retrovirology 7, 10 (2010).
6.Van Kuppeveld, F. J. et al. Br. Med. J. 340, c1018 (2010).
7.Lo, S. C. et al. Proc. Natl Acad. Sci. USA 107, 15874-15879 (2010).
8.Switzer, W. M. et al. Retrovirology 7, 57 (2010).
9.Robinson, M. J. et al. Retrovirology 7, 108 (2010).
10.Oakes, B. et al. Retrovirology 7, 109 (2010).
11.Sato, E. , Furuta, R. A. & Miyazawa, T. Retrovirology 7, 110 (2010).
12.Hué, S. et al. Retrovirology 7, 111 (2010).

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