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Germ wars
The Centers for Disease Control and
Prevention lab in Fort Collins is on the front lines of the war
against bioterror. Scientists are locked in a struggle with
life-or-death implications - preparedness and response to deadly
micro-organisms
By Jim Erickson, Rocky Mountain
News
August 24, 2002
FORT COLLINS - One of history's most
potent and feared killers dwells in a quadruple-locked freezer,
in the bowels of an aging federal laboratory tucked against the
foothills west of town.
Freeze-dried like instant-coffee crystals and sealed in more
than 1,000 tiny glass ampules, plague bacteria from around the
globe - culled from hapless human victims as well as rodents and
fleas - are stored in this boxy, nondescript beige Centers for
Disease Control and Prevention building.
Ducks squawk on the pond next door and pricey new custom
homes sprout from the hillside a few hundred yards away. It's
the last place anyone would describe as a war zone. Yet the
researchers inside these tightly sealed laboratories are, in
fact, front-line soldiers in this nation's war on bioterrorism.
Plague is on the CDC's list of "agents of highest concern"
for bioterrorism, along with the organisms that cause anthrax,
smallpox, tularemia, botulism and Ebola hemorrhagic fever. Used
as bioweapons, any of these highest-priority agents could cause
enough disease, death and disorder to cripple a large city.
It is the Fort Collins lab's job to monitor naturally
occurring plague - 10 to 15 cases are reported in this country
each year and 1,000 to 3,000 worldwide - and to spot unusual
clusters that could signal an intentional release.
The lab, which opened in 1967, is known as the Division of
Vector-Borne Infectious Diseases. Researchers there specialize
in the study of pathogens transmitted by mosquitoes, ticks and
fleas.
In 1999, bioterrorism preparedness was added to the lab's
mandate. In addition to plague, the Fort Collins lab tracks two
less-well-known but worrisome bioweapon candidates - tularemia
and Venezuelan equine encephalitis.
The main bioterrorism threat for all three microbes would be
inhalation of an aerosol germ cloud released into a building or
over a city.
"BT" projects fast-tracked
After Sept. 11 and last fall's anthrax attacks through the
mail, several pressing "BT" research projects at the Fort
Collins lab were fast-tracked by CDC headquarters in Atlanta.
And President Bush's budget proposal for the coming fiscal year
includes $74 million to build a new, bigger Fort Collins CDC
lab, which would mean more research on bioterrorism defenses and
insect-borne diseases.
"BT went from a high-priority program at CDC to a very, very,
very high priority - the top priority," said Duane J. Gubler,
director of the Fort Collins lab. "And since we work with three
of the top 10 potential BT agents here in this division, that
put a lot of focus on this program.
"I think the thing that Sept. 11 and the events following
Sept. 11 did is it drove the point home that we damn well better
be serious about it."
The highest-priority BT research projects at the Fort Collins
lab include efforts to develop new diagnostic tests for
emergency-response personnel, health officials and criminal
investigators.
Researchers there are at the forefront of an emerging
discipline known as microbial forensics, which uses tiny genetic
variations between bacterial strains to provide clues about the
source of bioterrorism agents.
In collaboration with researchers at several universities and
national laboratories, CDC scientists in Fort Collins are
working on:
• High-resolution genetic fingerprinting tests to
quickly trace the origin of intentionally released plague and
tularemia. In the next two years, Fort Collins scientists plan
to genetically map each of the more than 1,000 plague strains
and the roughly 200 tularemia strains in their collection.
• Techniques to quickly determine if plague and
tularemia bacteria were altered by terrorists to make them more
virulent or resistant to antibiotics.
• "Live organism detection" tests so first responders
can tell if intentionally released plague and tularemia bacteria
are alive and infectious - and therefore a genuine threat to
public health - or dead bugs that could trigger public panic but
pose no health risk.
Killer-germ elite
When health officials discuss the potential bioweapons of
greatest concern, plague is near the top of everyone's list,
often right behind anthrax and smallpox. Several factors combine
to place plague among the killer-germ elite, including its
fearsome history as a scourge of humanity.
Plague was the Black Death and the Great Pestilence that
wiped out 20 to 30 million Europeans - between one-quarter and
one-third of the continent's population - in the Middle Ages.
Even today, when antibiotic therapy and public health advances
make a global pandemic unlikely, the mere mention of the word
plague stirs a deep-rooted dread that terrorists could exploit
to foment panic.
Plague was first used as a weapon in the 14th century, when a
Tatar army catapulted diseased corpses over enemy walls in
Crimea. In World War II, a secret branch of the Japanese army,
Unit 731, reportedly sprayed grain laced with plague-infected
fleas over Chinese cities, causing outbreaks that killed
hundreds - perhaps thousands.
The Soviets took plague weapons to the next level during the
Cold War, eliminating the need for the unreliable flea by
perfecting an aerosol form of the germ.
Several common antibiotics kill naturally occurring plague
bugs. But in his 1999 book Biohazard, Ken Alibek, former deputy
chief of the Soviet germ-warfare program, said his scientists
developed genetically altered strains of plague "sufficient to
overcome practically all antibiotic treatments."
It would be easier for terrorists to acquire plague than
smallpox, a virus that isn't found in nature. The bacterium that
causes plague, Yersinia pestis, is studied in laboratories
across the globe and can even be isolated from infected wild
rodents, such as Colorado prairie dogs.
Resistant microbes
At the same time, the techniques for transferring
antibiotic-resistant genes into bacteria such as Yersinia pestis
are taught in introductory-level college microbiology courses.
And many of the tools needed to do so can be purchased cheaply
on the Internet, said May Chu, chief of the diagnostic and
reference section of the Fort Collins lab.
"One of the things we worry about is that somebody who has
the intent to create harm might engineer an antibiotic-resistant
gene so that it renders the treatment we recommend inadequate,"
Chu said.
An antibiotic-resistant plague weapon would be a medical
nightmare, though some researchers doubt that terrorists could
fashion one capable of defeating all the drugs that can kill
Yersinia pestis.
Compounding concerns over antibiotic resistance is the fact
that no existing plague vaccine is effective against pneumonic
plague, the lung-infecting form that would occur after an
intentional aerosol release. Pneumonic plague is contagious and
kills nearly all untreated victims.
Bubonic plague, the most common form of the disease, is not
contagious and has a lower kill rate. Several bubonic plague
vaccines exist, though none of them are currently licensed in
the United States.
Plague profiling
At the Fort Collins lab, Chu and her colleagues are compiling
a database containing genetic profiles of various plague
strains. Using these so-called "plasmid profiles," researchers
could quickly determine if intentionally released plague was
altered to make it more virulent, more environmentally resilient
or resistant to antibiotics.
"With these tests, we'd be able to tell you right away
whether it's been tampered with or not," Chu said. "And at one
quick look, we can tell if it's of U.S. origin or not."
The easiest way to transfer antibiotic-resistant genes into
plague and tularemia cells would be to load them inside
microscopic rings of DNA called plasmids, Chu said. Plasmid
transfer is a central tool of modern molecular biology and
microbiology, a technique that helped spawn the biotechnology
industry in the 1970s.
Chu launched the Fort Collins plasmid-profiling project in
1994 as part of a larger effort to study bacterial genetics. But
the program took on new meaning and added urgency after Sept.
11, and the emphasis shifted to forensic applications.
"The focus of my career has changed," Chu said.
"Now, I'm much more in the mind-set that everything I do has
some kind of bioterrorism preparedness and response connection.
The question you run through your head is, 'How would this
help?' "
The human body is built from about 60 trillion cells, and
DNA's genetic blueprint is stored on 23 pairs of chromosomes in
the nucleus of each cell.
The plague bacterium is a single-celled organism with a lone
chromosome that holds more than 99 percent of its DNA.
The remaining bits of plague's genetic material are in its
plasmids, rings of DNA less than one-twentieth the size of the
chromosome. Plague cells normally carry multiple copies of three
types of plasmids.
To create a plasmid profile, scientists analyze the number
and size of these DNA rings. Millions of bacterial cells - each
so small that 1,000 of them placed end to end would span the
head of a pin - are broken apart so the plasmids can be
separated from the chromosomes.
Then the plasmids are placed in a gel-filled tray and
subjected to an electrical current that separates them by size.
A special dye is used to light up plasmid DNA within the gel,
then a photo is taken of the distinctive banding patterns - the
plasmid profile - within the gel.
If plague is intentionally released, plasmid profiling could
be used to compare germ samples collected from a victim, or from
the release site, to CDC's computerized plague database. It's
similar to lifting a human fingerprint from a crime scene, then
searching records for a match.
"If we were to look at a plasmid profile of an isolate and we
find that there's an additional plasmid, that should raise some
alarm because most antibiotic resistance that could be
engineered intentionally is really by a plasmid," Chu said.
"And if you could pluck out that plasmid - which you can -
it's like a bomb signature, and you could probably find out then
how it got there, and you could probably then trace it back to
where it came from."
Microbe sleuths
Plasmid profiling is one of several techniques being plied by
a cadre of scientists in the field of microbial forensics, which
has been gaining momentum since last fall's anthrax-letter
attacks. In microbial forensics, small genetic variations
between strains of a microorganism are used to make inferences
about the origins of a particular isolate.
A central goal of this rapidly evolving discipline is to
develop microbial tests that are reliable enough to stand up in
court, said Abigail Salyers, past president of the American
Society for Microbiology. In June, a small group of
microbiologists and law enforcement officials met in Vermont to
chart a course for microbial forensics.
"This is the time that we should be thinking ahead, rather
than waiting until something happens and then trying to rush
around and figure out what we're doing," Salyers said.
"The FBI is interested in this, and they're just beginning to
apply this kind of molecular biology forensic analysis to the
materials they have. This is not something they're used to."
Salyers said it's vital to be able to quickly detect
terrorist-altered germs. But she stressed that creating and
delivering an antibiotic-resistant plague weapon would be "a lot
more difficult than it sounds."
"I don't doubt the Soviets were capable of doing this, but
the bacterium that causes plague is susceptible to a number of
different antibiotics.
"So it's hard for me to believe that they could have made
something resistant to everything," said Salyers, a professor of
microbiology at the University of Illinois.
"I think we should face the problem of antibiotic-resistant
bacteria, but I also think it's important for people to
understand that this is not something that you could just go and
do easily in any lab in the country, or in your garage, or
wherever."
Fingerprinting project
With funding from the U.S. Department of Energy, Fort Collins
CDC researchers are working on a second bioterrorism genetics
project called microbial fingerprinting. It could help
investigators find the source of intentionally released plague
or tularemia.
The Fort Collins researchers are collaborating on the
fingerprinting project with Northern Arizona University's Paul
Keim, who has played a key role in the anthrax-letter
investigation.
Working with the Institute for Genomic Research in Maryland,
Keim's Flagstaff lab has studied the complete genetic sequences,
or genomes, of various anthrax strains in an attempt to find
subtle variations that will help investigators nail the culprit.
During the next two years, CDC researchers in Fort Collins
plan to use similar techniques to create computerized genetic
fingerprints for all their plague and tularemia strains. The
database will be shared with the FBI, the Defense Department and
other agencies likely to respond to a plague or tularemia
attack.
Microbial fingerprinting uses many of the same
molecular-biology tools that are applied to human DNA samples in
paternity cases and in criminal cases such as the O.J. Simpson
trial.
In the CDC plague project, researchers will be looking for
distinctive patterns of repetitive DNA sequences in the Yersinia
pestis genome, which was decoded by British scientists last
year.
The plague genome contains the blueprint for all the tasks
the microorganism must perform and those instructions are
encoded in a series of chemical letters known as A, G, T and C.
The plague genome is more than 4.6 million letters long. The
Fort Collins researchers and their Arizona colleagues will look
for repeats, or stutters, in the sequence of words formed by the
four-letter genetic alphabet. The repeating patterns - somewhat
like multiple copies of a bar code - can be used to distinguish
between various bacterial strains.
The hope is that the distinctive patterns, called variable
number tandem repeats or VNTRs, could be used to quickly match
an intentionally released microbe to a strain in the CDC
database.
"The end product you hope for is the identification of a
strain to the point where you know exactly where it came from,"
Chu said.
In a related project, the Fort Collins scientists are working
with Lawrence Livermore National Laboratory to decode the
tularemia bacterium's genome.
Swedish and British researchers have nearly completed the
genome for the disease-causing strain of tularemia. The Lawrence
Livermore and Fort Collins scientists will decode the strain
used in tularemia vaccine and the two genomes will be compared.
The results should help identify targets for future vaccines and
drug therapies.
Living or dead?
The third major bioterrorism test being developed in Chu's
Fort Collins lab is called live organism detection. Unlike hardy
anthrax spores, which can survive for decades in soil, plague
and tularemia bacteria normally perish quickly when exposed to
sunlight or dry air.
Current tests can spot plague or tularemia bacteria in the
environment or at a crime scene. But no existing test can
quickly determine if those bugs are alive, Chu said. Growing
colonies of plague bacteria in the lab is one way to test for
live samples, but that takes at least a day.
At the Olympic Games in Salt Lake City in February, security
teams set up about a dozen portable laboratories to analyze air
and environmental samples for various biological and chemical
agents. If one of those tests had come back positive for plague
during opening ceremonies at Olympic Stadium, for example,
health and law enforcement officials would have faced a
momentous decision.
"OK, so you have a positive signal. Is it a threat or is it
not a threat?" Chu said.
"If you had a test for live, viable organisms, you can
quarantine right away and limit the spread," she said.
"If it's replicating, then we're in trouble," Chu said.
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