A not-so-new threatthe West Nile virus
In 2002, there were some 3,500 U.S. cases of West Nile virus.
IN 1937, A WOMAN FELL ILL in Ugandas West Nile Valley, and scientists identified the causea brand-new virus that soon began to spread, picking up speed and scope as it went. By the 1990s, this West Nile virus was popping up in Romania, parts of the former Soviet Union and Israel, where it affected geese and humans. Late in the summer of 1999, it reached the United States, sickening 62 people in New York, killing seven, and devastating the crow, blue jay and hawk populations. Since then, it has moved swiftly south and west, and in 2001, it reached the Midwest.
In 2002, there were some 3,500 U.S. cases of West Nile virus161 in Missouri and 741 in Illinoiswith as many as 200 deaths. In many places, the skies have grown more quiet as birds have fallen victim. Science magazine estimated recently that as many as 100,000 crows may have died of the disease through this past summer and early fall. Horses, rodents and other birds, including zoo species, also have been affected.
This year was a very big one for transmission, with West Nile moving all the way to the states of Washington and California, says Michael S. Diamond, MD, PhD, assistant professor of medicine, molecular microbiology and pathology and immunology. By next year, it will be in every one of the 48 contiguous states, then we expect it to move south into Mexico, very soon hitting Central and South America.
The widening impact of the virus has sparked a wave of scientific detective work. The Centers for Disease Control and Prevention (CDC) has been charting its progress; so has the National Institutes of Health (NIH), which has labeled the West Nile virus a priority organism. And a handful of infectious disease researchers are studying the molecular roots of the disease, hoping that this understanding will translate into effective new agents for its prevention and treatment.
Michael Diamond is one of them. Since joining the School of Medicine faculty in July 2001, he and his research teamassociate Mike Engle, postdoctoral fellows Bimmi Shrestha and Brian Geiss, graduate student Erin Mehlhop and technician Anantha Marrihave been targeting West Nile virus, asking a series of questions. On a cellular level, what does the virus attack and how? Is a piece of the immune system malfunctioning, keeping mice and humans from fighting the virus? And if, as they now believe, the answer is a particular antibody, will administering that antibodyeither in the form of pooled gamma globulin or humanized monoclonal antibodieshelp prevent humans from getting the disease or help those who are already ill?
Only one in 100 people bitten by a disease-bearing mosquito will become sick enough to go to the hospital; 70 percent never have symptoms at all, while 20 percent have mild, flu-like disease. From epidemiological studies, the team also knows that people over 50 are at increased risk for the disease, as are those with compromised immune systems, such as transplant, HIV, cancer or kidney failure patients. But why are some affected while others are not? And why is age an evident risk factor?
Diamond and his group are actively pursuing these questions, spending 80 percent of their time on West Nile. Backed by grants from the NIH, CDC, Pharmacia and the Ellison and Mallinckrodt foundations, they do their work in a special laboratory containment facility with specific air pressure and security requirements. And they draw expertise from collaborators in related areasinfectious disease, immunology, virology and neurobiologywho have helped them get started with their research.
At first, the group established a mouse model in immunocompetent mice, inoculating them with the virus by injecting it, mosquito-style, just under their skin and watching the infection disseminate. The disease process in these mice closely mirrored what appears to occur in humans. After a few days, the virus moved to their spleens and lymph nodes, then into their brains and spinal cords. But just as in people, only a small number got severely ill, showed evidence of paralysis or died.
In examining the brains of these mice under a microscope, researchers saw that the virus had damaged their neuronsbut why? To study this question, they obtained mouse embryonic stem cells and embarked on the difficult task of differentiating them into neurons. And they found that, while the stem cells themselves were resistant to West Nile infection, the neurons were easily infected and quickly died.
That suggests, says Diamond, that the virus is actually getting into the cells themselves, replicating and causing injury directly. So one part of our lab has begun to try to understand what the mechanism of injury is in the neuron.
Some mice did not die; instead, they became immune to the disease. Why were they able to combat the virus effectively, while others were not? If we take away a certain piece of the immune system, the researchers wondered, will that prevent the mouse from combating infection? To look further, they began working with genetically engineered knock-out strains of mice that are genetically identical except for one specific aspectsuch as a particular T or B cellof immune system function.
When they used mice deficient in B cells and antibody, those mice became extraordinarily susceptible to disease. Even when they received only minuscule doses of virus, 100 percent got sick. This finding, they speculate, could shed some light on why older people are more likely to be affected by West Nile virus, since studies have shown that as people age, their antibody function changes and antibody dysfunction may even set in.
In the mouse model, we know that if you dont have antibodies you are in big trouble, Diamond says. In people over 50, a subset may have dysfunctional antibody responses, and those people could be more likely to have a disseminated West Nile infection that goes into their brain.
Next, the researchers began pre-clinical trials in mice: taking serum containing antibody from wild mice who survived infection and were now immune to West Nile virus, and giving that antibody to B-cell-deficient, knock-out mice. The serum protected those deficient mice completely, making them entirely resistant to West Nile infection.
How would that work in humans? With the West Nile outbreaks in Israel, some people there have developed immunity to the virus and have antibodies in their blood streams; when they donate blood, those antibodies go into the pooled blood supply used to make gamma globulin. A year ago, an Israeli woman, sick with West Nile, received a shot of gamma globulinand recovered from the disease. One report is far from conclusive, but to Diamonds team it was suggestive.
So they have obtained Israeli gamma globulin and tested it in both the normal and B-cell deficient mice to see if it prevents them from becoming ill with West Nile virus. The results have shown that it has been protective, though the concentration of antibodies in the serum is rather low. As a next step, they are beginning to test gamma globulin as a therapeutic agent, used to cure mice that have fallen sick.
Ideally, says Diamond, the researchers would like to have a less diluted source of antibody, and one way would be to create monoclonal antibodies from mouse cellspurifying several antibodies that could be combined into a potent antibody cocktail that may work better than gamma globulin. If it does, those mouse antibodies would then have to be genetically humanized for use in people.
But the monoclonal antibody phase of their work is just beginning, he cautions. Even if it goes smoothly, human agents from this work are still several years away. But it is possible that they could some day be used as a short-term way to prevent the most susceptible population from getting the virus, and possibly as therapeutic agents once people are infected.
I believe that immunotherapeutics may have some basis for prevention or treatment, says Diamond. It looks promising, and we are optimistic, but in science you can do 10 experiments and have most of them not work, so we will have to see how these studies go before we know for sure.