William Romoser still has fond memories from a weekend spent camping along a lake in northern Ontario at the impressionable age of 6. The Ohio University professor of entomology remembers the taste of the blueberry tarts and glimpses of fish and turtles, but he also recalls one unmistakable sound: the buzz of mosquitoes. Romoser spent the night in his lean-to being harassed by the irritating insects.

“I didn’t know at that time I was going to study them someday,” Romoser says with a laugh. The scientist has been dedicated to studying these insects since he was a graduate student at Ohio State University in the 1960s, and few people know better than he does that these insects are more than just irritating: They can pose severe health problems. Currently, Romoser is interested in how arboviruses, a major cause of human encephalitis (inflammation of the brain), travel through mosquitoes and eventually infect humans. “The basic question now is: Why do some mosquitoes transmit viruses and others don’t?” he says.

It’s a good question considering that thousands of people inthe United States are infected with an arbovirus annually.Mosquitoes primarily transmit arboviruses, but not all bites areequal. Different mosquitoes carry different diseases, and not allinfected mosquitoes transmit their diseases to humans. Speciesdiffer in their feeding patterns, preferred hosts, and the placesthey choose to lay their eggs. A mosquito’s ability to pass alonga disease is called its vector competence, and understandingthis concept is key to Romoser’s research. “There are 3,500plus mosquito species in the world and over 60 in Ohio,” heexplains. “Each has its own lifestyle, so to speak.”

Infectious Concerns

The public has become warier of mosquitoes since the highly publicized outbreak of West Nile virus began in 1999. In 2003 alone, 8,734 cases of the virus and 208 deaths were reported to the Centers for Disease Control and Prevention (CDC), according to statistics on the organization’s Web site in December. But West Nile isn’t the only mosquito-borne illness of concern to public health officials and scientists such as Romoser who study the way such diseases are transmitted.

The cattail marsh mosquito, for example, is one of the species known to spread Eastern Equine Encephalitis. In humans, this disease can range from mild flu-like symptoms to seizures or a coma, according to the CDC. While the West Nile virus has a mortality rate of up to 15 percent, Eastern kills 30 percent of those infected with the disease. In 2003, the CDC began reporting cases of Eastern Equine Encephalitis on ArboNet, a national electronic surveillance system established to assist states in tracking West Nile virus and other mosquitoborne viruses, according to the organization’s Web site.

For years before West Nile made headlines, Romoser had been studying diseases related to West Nile and Eastern Equine Encephalitis, the latter of which is a domestic version of some of the predominantly foreign viruses he studies. Some of the findings from his studies most likely will be applicable to understanding Eastern Equine Encephalitis, he says. With colleagues at the University of California-Davis, Romoser has been examining the transmission process of St. Louis Encephalitis, a close relative to West Nile that has been found in Ohio, the Gulf Coast, and the Mississippi Valley, and Western Equine Encephalitis, which, as its name suggests, is found in the western United States.

The scientist also has been collaborating with the U. S. Army on studies of Venezuelan Equine Encephalitis, a predominantly South American and Central American disease that has appeared in pockets of the United States, and Rift Valley fever, an African virus that has potential to occur here as well. The Army has identified several domestic mosquito species that could transmit Rift Valley to humans and animals, he explains.

Though some of those diseases may be thousands of miles away from home, Ohio has documented four mosquito-borne arboviruses to date: LaCrosse Encephalitis, St. Louis Encephalitis, West Nile, and Eastern Equine Encephalitis. Among these, Eastern Equine Encephalitis is especially dangerous.

“It’s really a nasty disease,” says Robert Restifo, chief of the Ohio Department of Health’s Vector-borne Disease Program.“Those that do survive often have serious neurological damage.”

The severity of this disease definitely puts it on the department’s radar, along with the fact that two counties in Ohio — Wayne and Holmes — are now identified as “hot spots” due tomarshy regions there. But the areas in Ohio are rare for thisdisease, which usually occurs along the Gulf and Atlantic Coasts,due to the virus’ need for such a specific marshy area, Restifo says.

The stream of mosquito-related facts seems to come effortlessly for Restifo, who has worked for the program since 1975. The official also knows the benefits Romoser’s research could bring to the public health spectrum.

“Bill’s research will help us define which species of mosquitoes are good vectors of these diseases,” he says. “If we know what species are capable of transmitting a particular disease, and we know we have activity of that disease in the area, then we can concentrate on those habitats,” he adds. “It makes the whole control issue more effective and efficient.”

Road Trip

Romoser plucks a slender test tube from a box and tilts it slightly, examining the black speck floating inside. “This is how the mosquitoes arrive at the lab,” he says. The perfectly preserved mosquito has taken quite a trip before arriving at Romoser’s laboratory at Ohio University’s Irvine Hall, where he serves as professor of medical entomology for the College of Osteopathic Medicine, and the work is far from over.

About 450 miles away in Ft. Detrick, Maryland, scientists at the United States Army Medical Research Institute of Infectious Diseases infect the mosquitoes with the viruses. Mosquitoes are fed infected blood and then held for several days to two weeks,says Lee Wasieloski, a scientist at the institute who also is interestedin virus and host interactions.

“We then test them for viruses at different time points and try to determine whether the whole mosquito is infected and whether or not the salivary glands are infected,” he says. Groups of about 10 mosquitoes are collected and preserved at different times, Wasieloski says. Then they are ready to make the long trip to Athens, Ohio.

“This is our fancy bologna slicer,” Romoser says with a smile, pointing to a metal contraption perched on the laboratory counter. Surprisingly, the instrument wouldn’t look out of place in a local deli, but this “slicer” serves a more scientific purpose than the creation of the perfect sandwich. It takes one mosquito and transforms it into a visible timetable of disease no bigger than a stick of gum.

To prepare the mosquito, Romoser embeds the insect in a square of paraffin, a type of wax. “It gets all through the tissues and makes the mosquito firm,” he says, “so now we can cut it.” The small block of wax is placed on the slicer and Romoser cranks the device. A ribbon of paraffin starts to emerge, and it’s dotted with thin sections of the mosquito.“It’s like pages of a book spread out,” Romoser says. “You have a whole mosquito on a microscope slide.”

Then the mosquito is ready to divulge its medical history to Romoser. He sends in his “bloodhounds” to find the virus. These are antibodies that are dribbled directly ontothe mosquito sections and seek out the virus, wherever it is.A second antibody is then sent in to track down the first.This secondary antibody is paired with a “tag” or moleculethat will ultimately make some type of visible reaction. “Thetag could either be something that glows when you shineUV light on it,” Romoser says, “or it can be a chemical thatcauses a color reaction.”

The slide is then dehydrated and a cover slip is mounted on it to create a permanent record Romoser can look at now or a year from now and still see the path of the virus. Romoser views the slide on a microscope with a digital camera, which then transmits its photos to a computer screen. On one slide, striated stomach muscles glow eerily green with infection.

Lock And Key

Over the years, the scientist has made some interesting observations: He’s found some instances when the mosquito takes a virus into its stomach, and the stomach cells fail to become infected. He calls this an “infection barrier,” and while this may or may not be a physical block, the inability to infect could be associated with the absence of a receptor molecule on the cell. It’s similar to a lock and key arrangement. “Particular viruses infect particular cells because the cells have the right lock and the virus has the right key,” he says. The presence, absence, abundance, or accessibility of certain receptor molecules on the surface is one of the keys to infection, he says.

There is one specific infection barrier that Romoser and colleague Michael Turell at Ft. Detrick have found in Anopheles stephensi, a mosquito species that transmits malaria in Asia and serves as a good laboratory model for the arboviruses, Romoser says. In his research, he’s found that the mosquito’s basal lamina, the noncellular layer surrounding its salivary glands, appears to prevent the virus from infecting these glands, the last stop on the viral path. Avoiding salivary gland infection is important because once the virus is detected in the glands, it’s a reasonably safe assumption that the mosquito’s next bite has disease-causing potential, Romoser says. “If the virus can’t infect the mosquito, it can’t transmit it,” he adds.

Mosquito Mosaic

But there’s a lot more to discover in the field of medical entomology, and Romoser and his colleagues view the project as an ongoing process. The end result will be a “mosaic” of various scientists’ work, he notes. “The ultimate goal is to understand the mechanism and find a way to intervene and prevent virus transmission,” says Romoser, whose most recent vector competence findings will be published in the Journal of Medical Entomology.

Romoser’s fascination with the mysteries of the anatomy of mosquitoes and the diseases they transmit will keep him engaged in the project for many years to come. His mosquito studies have taken him around the world on field expeditions much larger than that first camping trip in Ontario. “You like to feel that you are working on something of importance and making a contribution to social welfare and public health,” he says, “but fundamentally it’s curiosity that drives you.”

For more information about Romoser’s research, visit
www.oucom.ohiou.edu/dbms-romoser/.