Military Bug Chasers Help Track Down West Nile Virus

By Sgt. 1st Class Kathleen T. Rhem, USA
American Forces Press Service

FORT DETRICK, Md., May X, 2001 - A small team of
scientists on this sleepy base north of Washington played a
crucial role in tracking down one of the more visible
public health threats to hit North America in the past
decade. And they're still working to make it easier for
public health departments to fight the disease.

Scientists at the U.S. Army Medical Research Institute of
Infectious Diseases here worked closely with the federal
Centers for Disease Control and Prevention and the U.S.
Department of Agriculture to identify the West Nile virus
when it first appeared in North America. In 1999, the virus
killed seven people and countless birds in and around New
York City. It has since spread up and down the East Coast.

West Nile virus was first identified in 1937 in Uganda.
Today its range sweeps from the southern tip of Africa
through southern Europe, southwestern Russia and east as
far as India and Pakistan. It generally causes low fever
and very mild cold symptoms in people, but can kill the
very old or sick.

In summer 1999, Tracey McNamara, a veterinary pathologist
at the Bronx Zoo, became concerned when birds began dying
in the zoo, both rare, zoo-collection birds and common
native crows, said George Ludwig, the Army institute's
chief of applied diagnostics. The CDC tentatively
identified a nearby outbreak of human illness as St. Louis
encephalitis, which is caused by a virus similar to West
Nile.

"But birds don't die from St. Louis encephalitis," Ludwig
said. So McNamara didn't buy that diagnosis.

"The pattern of the types of birds that were dying didn't
fit with known viruses," said Army Maj. Tom Larsen, the
institute's chief of experimental pathology. "There are
other diseases along the Eastern seaboard that will cause
death in birds, but this wasn't causing death in the right
kinds of birds."

The quest to identify the mystery virus was on.

It didn't take long for USAMRIID scientists to identify the
unknown virus as a flavivirus, which includes West Nile,
but also St. Louis encephalitis, Japanese encephalitis,
dengue and yellow fever. That's when things slowed down.

According to Ludwig, tests available at the time would have
reacted to any flavivirus. It took a long, difficult
process of genetic sequencing to positively identify West
Nile virus.

No one was more surprised than the researchers working on
the project when West Nile virus turned up. "West Nile had
never been seen anywhere in North America before 1999,"
Ludwig said.

USAMRIID scientists have since developed two distinct tests
that can specifically identify West Nile virus without
cross-reacting with related viruses, Larsen said. One
identifies specific virus proteins; the other seeks out
nucleic acids.

Before the development of these tests, only a few
laboratories in the country could positively identify West
Nile virus. Now, thanks to the work of USAMRIID scientists,
many more laboratories have the capability.

In the first method, immunohistochemistry, perfected for
West Nile virus by the USAMRIID pathology staff, antibodies
are introduced into a tissue sample suspected of being
infected with the virus. If virus is present, the
antibodies attach themselves to proteins in the virus. This
reaction is invisible, even under a microscope.

The addition of a second antibody with a specific enzyme
attached sets up conditions for the final test step: the
addition of a chemical that changes colors if the viral
proteins are present.

The other testing method, in situ hybridization, is
similar. "Instead of looking for viral protein, you're
looking for nucleic acid," Ludwig said. He explained that
genetic material is made up of four nucleotides. Two of the
nucleotides complement the other two, which allows them to
bind together.

Scientists had to design a specific sequence of nucleic
acid that will bind only to the genetic material of the
West Nile virus. "This primer is connected to an enzyme
that, in the presence of another chemical, produces a
colored reaction," Ludwig said.

USAMRIID has worked to make both methods available to other
scientists. That in turn will make it easier for public
health departments to control the spread of the virus.

But both methods have limitations, Larsen said.

It's relatively easy to make the in situ hybridization test
available to others, Larsen said. You simply publish the
correct genetic sequence and other labs make what they need
or order it from a supply company.

"But in situ hybridization is very time-consuming," Larsen
said. "And very few people do it."

Immunohistochemistry is much easier and quicker, he said,
but it's harder to obtain the antibodies needed to complete
the test. "It takes special lab capabilities to make
antibodies that are monoclonal, meaning they react only to
a specific substance," Ludwig explained. "Many antibodies
cross-react with any similar substance."

Ludwig said USAMRIID helps make the monoclonal antibodies
available to others through a cooperative research and
development agreement with BioReliance Corp., a local
civilian company.

"They are producing the antibody to supply to people," he
said. USAMRIID supplies the antibodies to other labs within
the federal government. "We send them some live cells, and
they can grow the antibodies themselves," Ludwig said.

USAMRIID also serves as DoD's West Nile virus reference
center. Whenever a DoD medical asset has a person they
believe might be suffering from West Nile virus, they send
samples here. Ludwig estimated his team has tested about 30
samples in the past two years from bases along the East
Coast. All were negative, he said.

If fighting a disease outbreak is like putting together a
puzzle, then identifying the culprit is only a small piece.
Ever since the virus was identified, USAMRIID scientists
have been trying to determine how it spreads from one area
to another and from birds to people.

They knew from studies in other parts of the world that
West Nile virus is mosquito-borne, but as far as prevention
goes, that presents more questions than answers, according
to USAMRIID entomologist Michael J. Turell.

Mosquitoes are not created equal, Turell said. "Certain
mosquitoes transmit certain pathogens and not others.
Certain mosquitoes breed in standing water, others breed in
tree holes, in empty tires or in streams," he said. "Some
are day biters, and some are night biters."

Turell said scientists did not know in 1999 which of the
hundreds of varieties of North American mosquitoes spread
the virus. Scientists can test mosquitoes from an area to
determine if they carry the virus, but "carry vs. transmit
is a very fine point," he said.

Mosquitoes are tested by freezing then grinding them up.
West Nile virus is present three different ways in mosquito
bodies, Turell said. It can be in a blood meal the insect
just ingested; the mosquito itself can be infected; or it
can be ready to transmit the virus to a person or other
animal. Then, too, not all infected mosquitoes can transmit
the virus.

"If a mosquito feeds on a West Nile virus-infected animal,
the blood in its gut contains virus and the gut might
become infected," Turell said. "But a mosquito with an
infection limited to its gut cannot transmit virus. The
virus has to get out of the gut, through the mosquito's
body and into the salivary glands. Then it can transmit."

After a mosquito has been ground up, there's no way to tell
where the virus was inside its body. "Just saying a
mosquito was carrying virus in its body doesn't mean this
mosquito is a health threat," Turell said.

To determine which breeds of mosquitoes are competent
carriers, scientists allow mosquitoes to feed on infected
birds and wait two weeks, the usual timeframe for the virus
to move through the mosquito to its salivary glands. The
mosquitoes are then allowed to feed on non-infected birds,
to see if the birds become infected.

Even knowing which mosquitoes can transmit the virus is
only another small piece of the puzzle. If the mosquitoes
don't feed on the right species, primarily birds and
humans, they still aren't a public health threat.

West Nile virus primarily lives in crows, which it kills,
and house sparrows, which it doesn't. USAMRIID scientists
have identified several members of the Culex genus
of mosquitoes as responsible for spreading the virus among
birds. However, Culex mosquitoes rarely feed on
people, Turell said.

"Many North American mosquitoes are much less fussy about
what they feed on," he said. "I've seen salt marsh
mosquitoes try to feed on warm automobile tires." He said
some breeds may not be efficient carriers, but they pose a
health risk because they occur in large numbers and feed on
both birds and people.

"A recently introduced mosquito species, Ochlerotatus
japonicus, is spreading down the East Coast and is of
particular concern," he said. "It is an efficient
transmitter of West Nile virus in the lab and has been
found naturally infected with this virus.

Humans rarely produce enough virus in their blood for
mosquitoes to transmit it to others, Turell said.

Another piece of the puzzle is knowing the breeding and
feeding habits of mosquitoes that are efficient carriers.
This helps the public health or public works officials
responsible for controlling mosquitoes best target their
eradication programs. If a targeted mosquito only feeds at
night, spraying during the day would be a waste of time,
for instance.

It also helps to let people know what situations to avoid
if West Nile virus is prevalent in an area.

"By knowing the habits of the mosquitoes, we can say, 'You
know, going to Little League games after dusk could be
really dangerous. But up until dusk, there's no risk,'"
Turell said. "Or, 'Another species bites only during the
day, so canceling night games is meaningless.'"

Turell and his coworkers have shared what they've learned
with the CDC and with public health departments along the
East Coast and across the country. "They know which
mosquitoes they have in their area," Turell said. "When a
virus shows up in an area, they know which mosquitoes they
need to target."

He called the West Nile virus's spread to North America "an
effective wake-up call" for public health departments.
Their success in controlling mosquito-borne illnesses in
the past has led to steady cuts in public health and
mosquito abatement funding for "the last couple decades,"
he said.

"If they're doing their jobs, public health threats go down
and funding gets reduced for public health departments,"
Turell said. "But then the threat can increase again."

He said the West Nile virus's migration to North America
shows how easily even more serious health threats could
enter our country.

"Foot and mouth, Ebola, 'mad cow' - any of these could
come to the United States any day," Turell said. "The
public health infrastructure is critical in early
recognition and containment of many different health
threats. The question isn't if they're going to get here,
it's when they're going to get here. Air travel just makes
it too easy to move these things across borders."