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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 10, 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.

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West Nile Virus.
  

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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."

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U.S. Army Medical Research Institute of Infectious Diseases

Click photo for screen-resolution imageRoswita Moxley, a biological lab technician at the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Md., prepares slides of tissue samples to be tested for West Nile virus. Photo by Sgt. 1st Class Kathleen T. Rhem, USA.  
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Click photo for screen-resolution imageEntomologists at the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Md., raise several different species of mosquitoes for later testing to determine which can transmit the West Nile virus. Photo by Sgt. 1st Class Kathleen T. Rhem, USA.  
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Click photo for screen-resolution imageEntomologists at the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Md., raise several different species of mosquitoes for later testing to determine which can transmit the West Nile virus. Photo by Sgt. 1st Class Kathleen T. Rhem, USA.  
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Click photo for screen-resolution imageRoswita Moxley, a biological lab technician at the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Md., prepares slides of tissue samples to be tested for West Nile virus. Photo by Sgt. 1st Class Kathleen T. Rhem, USA.  
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Click photo for screen-resolution imageRoswita Moxley, a biological lab technician at the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Md., prepares slides of tissue samples to be tested for West Nile virus. Photo by Sgt. 1st Class Kathleen T. Rhem, USA.  
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Click photo for screen-resolution imageArmy Spc. Clark Dutterer, a preventive medicine specialist at the U.S. Army Center for Health Promotion and Preventive Medicine at Aberdeen Proving Ground, Md., sets a "Gravid trap" in the Maryland woods. Gravid mosquitoes are females that have recently had a blood meal and are ready to lay eggs. The mosquitoes are attracted to a substance in the trap, and a fan sucks them in when they get close. The mosquitoes are then tested to see if they are carrying certain diseases. Photo by Sgt. 1st Class Kathleen T. Rhem, USA.  
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Click photo for screen-resolution imageArmy Spc. Clark Dutterer, a preventive medicine specialist at the U.S. Army Center for Health Promotion and Preventive Medicine at Aberdeen Proving Ground, Md., sets a light trap to catch adult mosquitoes in the Maryland woods. The mosquitoes are then tested to see if they are carrying certain diseases. The trap was supplied by the Centers for Disease Control and Prevention. Photo by Sgt. 1st Class Kathleen T. Rhem, USA.  
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