# News this Week

Science  15 Oct 2004:
Vol. 306, Issue 5695, pp. 384
1. DRUG SAFETY

# Withdrawal of Vioxx Casts a Shadow Over COX-2 Inhibitors

1. Jennifer Couzin

On 30 September, the drug giant Merck announced that it was yanking its blockbuster anti- inflammation medicine, the COX-2 inhibitor Vioxx, off the market after an alarming pattern surfaced halfway through a 3-year colon polyp prevention study. Heart attacks and strokes had occurred at a much higher rate among the roughly 1300 volunteers on Vioxx (3.5%) than among the 1300 taking a placebo (1.9%). Within days, pharmacies were packing up their supplies of Vioxx and shipping them back to the company.

The scale of the withdrawal was unprecedented, casting a shadow over Merck, based in Whitehouse Station, New Jersey, and raising questions about the entire class of COX-2 inhibitors. Used primarily to treat arthritis and inflammatory pain, the drugs have earned billions of dollars since coming on the market more than 5 years ago. But the question could hardly be avoided: Did Vioxx collapse because of flaws unique to its chemistry, or would other COX-2 inhibitors suffer a similar fate?

“There are a lot of things we need to know now,” says Garret FitzGerald, a pharmacologist and cardiologist at the University of Pennsylvania in Philadelphia. “The game has shifted.”

Vioxx's propensity to trigger heart attacks and strokes isn't fully understood. But some experts believe that its valued mechanism—specifically, its ability to suppress a narrow set of molecules that mediate inflammation—may have been its downfall. Targeted drugs are all the rage, but many scientists worry that this particular targeting can upset a delicate balance that keeps blood-clotting at bay.

Drug regulators, among others, appear to be thinking along these lines. Last week, the European Medicines Agency in London said it would begin reviewing the safety of other COX-2 inhibitors, including Celebrex, made by Pfizer, based in New York City. U.S. experts at the Food and Drug Administration (FDA) and elsewhere cautioned against lumping other COX-2 inhibitors with Vioxx, but at the same time they have begun to review some studies of these drugs, including for pain, cancer inhibition, and Alzheimer's prevention. Richard Goldberg, chief of hematology and oncology at the University of North Carolina, Chapel Hill, learned for example that the National Cancer Institute and others overseeing his trial of Celebrex for preventing colon polyps, slated to enroll 1200 volunteers, were considering whether it might harm participants.

Manufacturers sought to reassure the public last week about their COX-2 products, a class that includes two Pfizer drugs on the market, Celebrex and Bextra, along with Prexige, made by the Swiss company Novartis, and Arcoxia, a Merck drug. The last two are approved in parts of Europe and are in late-stage development in the United States. Pfizer took a bold step, promoting claims of Celebrex's safety in full-page newspaper ads. But as some experts noted, studies of these drugs submitted to FDA did not last as long as the Vioxx colon polyp study. In that case, Merck didn't see serious problems until 18 months into the trial.

Celebrex was the first COX-2 drug, introduced in early 1999. Before that, arthritis patients relied mainly on nonsteroidal anti-inflammatory drugs such as aspirin and naproxen (marketed as Aleve) to blunt symptoms. In some patients, though, these drugs can cause stomach problems.

COX-2 inhibitors were hailed and heavily promoted as a major breakthrough because they home in on COX-2, an enzyme implicated in inflammation, while largely avoiding COX-1, which protects the stomach from gastric acids. Earlier anti- inflammatories had targeted both.

But preventing gastric upset may come at a cost. “Anyone who sits down with a pencil and paper and maps out the sequence of events” triggered by Vioxx “would have to say, ‘Could this enhance thrombosis?’” says Benedict Lucchesi, a cardiovascular pharmacologist at the University of Michigan, Ann Arbor.

The theory Lucchesi favors, which FitzGerald also endorses, is based on how two fatty acids work. One, prostacyclin, stops platelet formation and prevents the cells from clumping; it also dilates blood vessels. The other, thromboxane, has the opposite effect, encouraging platelet clumping and constricting vessels. Anti-inflammatory drugs like naproxen suppress both prostacyclin, which plays a role in inflammation, and thromboxane. But COX-2 inhibitors block only prostacyclin; this may tilt the balance in favor of thromboxane and, potentially, blood clotting. So far the thrombosis theory has been supported only by animal studies.

Still, the COX-2 drugs on the market are unique molecules and differ in critical ways. For example, they vary in how tightly they target COX-2 and avoid COX-1. They also vary in how long they linger in the body. Even if the thrombosis theory holds, the risk of blood clots from COX-2 inhibitors almost certainly differs from drug to drug. Vioxx is both highly targeted to COX-2 and has one of the longest half-lives, upward of 14 hours, a combination that some speculate may have triggered its problems. “We don't have a good explanation about why Vioxx is an outlier,” says Eric Topol, who heads cardiovascular medicine at the Cleveland Clinic in Ohio and, along with some other physicians, has long harbored concerns about the drug. “It's always carried the worst risk of heart attack and stroke, of blood pressure elevation, of heart failure.”

But some experts are not completely satisfied with the thrombosis theory. “I doubt that's the entire explanation” for Vioxx's dangerous effects, says Thomas Schnitzer, a rheumatologist and assistant dean of clinical research at Northwestern University in Chicago. Like all nonsteroidal anti- inflammatory drugs, Vioxx tends to boost blood pressure. Schnitzer wonders if this might be its Achilles' heel. Merck officials, however, told FDA that when they looked for a link between increased blood pressure and the heart attacks and strokes in the colon polyp trial, they didn't find one.

Topol, for one, believes more needs to be done: In an editorial released last week by The New England Journal of Medicine, he suggests that there could be “thousands of affected people” and calls for a congressional inquiry into Merck and FDA's handling of Vioxx in the years since it was approved.

2. INFLUENZA

# Crisis Underscores Fragility of Vaccine Production System

1. Martin Enserink

A snafu at a vaccine factory in Liverpool, U.K., has derailed U.S. plans to prepare for this year's flu season—and focused fresh attention on the fragile supply of essential vaccines.

Last week, Chiron, a pharmaceutical company based in Emeryville, California, announced that its Liverpool factory, which sells 90% of its vaccine to the United States, is unable to deliver any flu vaccine this year after British regulatory authorities effectively shut down the plant. The news sent U.S. authorities scrambling to ensure that the remaining vaccine supply—some 55 million doses, instead of the 100 million or more they had counted on—goes to those most at risk of complications and death, such as people over 65 years of age.

Chiron first reported on 26 August that its vaccines would be delayed because a small part of this year's batch of 50 million doses was contaminated with Serratia marcescens, a microbe that can cause opportunistic infections. Still, Chiron CEO Howard Pien assured a U.S. Senate Special Committee on 28 September that the company would eventually deliver 46 million to 48 million doses.

But on 5 October, the U.K.'s Medicines and Healthcare Products Regulatory Agency abruptly suspended Chiron's license to produce vaccines for 3 months, saying the company did not comply with so-called Good Manufacturing Practice regulations. Chiron, which acquired the plant last year when it bought the British company PowderJect, called the setback a “public health tragedy” but has declined to say how much of the vaccine is contaminated or what caused the problem. U.S. Food and Drug Administration (FDA) officials visited the plant in Liverpool last weekend to investigate. At a House hearing last week, acting director Lester Crawford appeared pessimistic that part of the batch might be salvaged.

The shortage comes at a time when a record 185 million Americans were advised to get flu shots. In guidelines issued this spring, the Advisory Committee on Immunization Practices (ACIP) had added children between 6 and 23 months and their close contacts to the list of groups that should get the vaccine. (It already included people over 50, patients with chronic illnesses, pregnant women, and nursing-home residents, as well as anyone who might transmit the virus to people in these groups.) After the Chiron announcement, ACIP pared down the list during a hastily convened meeting that same day, striking, for instance, parents of young children and healthy people between 50 and 65 and urging anyone not in a risk group to forgo the shots this year.

The number of people actually vaccinated is always much smaller than the recommended numbers, says immunologist Paul Offit of the Children's Hospital of Philadelphia, a former member of ACIP. Even so, the remaining lots—about 54 million doses of injected, killed vaccine from Aventis Pasteur, and 1 million to 2 million of FluMist, a live intranasal vaccine produced by MedImmune—will not be enough, Offit predicts: “There will be people who want the vaccine, who can't get it, and who will die because of that.”

Underlying the problem is an exodus of pharmaceutical companies from the vaccine business, which is widely seen as risky and not lucrative. The dwindling manufacturing base has led to previous severe shortages of some vaccines in the United States (Science, 15 March 2002, p. 1998). Production of the flu vaccine is especially vulnerable because its exact composition changes annually. Companies produce the vaccine between March and September every year in a tightly choreographed process. That's why no company can easily fill the gap left by Chiron, says David Fedson, a former medical director of Aventis Pasteur MSD who lives in Sergy Haut, France. The fragile supply could prove catastrophic should a new pandemic flu emerge, Fedson cautions (see p. 394).

Many solutions have been floated—from subsidizing companies to building a new government-operated vaccine plant—but little has been done. The current crisis should put the issue back on the agenda, says epidemiologist Arnold Monto of the University of Michigan, Ann Arbor: “We really need a sea change.”

3. MEDICINE

# Microbicide Shuts the Door on HIV

1. Jon Cohen

Microbicides have long had a stepchild status in the AIDS research community. Industry has had little interest in developing a topical gel or cream that can stop HIV at the vagina or rectum, and the products that have moved furthest in human studies are soaps and other substances that do not specifically target the virus. But over the past few years, nonprofits and governments have poured substantial money into microbicide research and development, bringing forward several cutting-edge concepts. On page 485 of this issue, an international team of researchers describes a monkey study that features one such strategy: a microbicide specifically designed to block HIV's ability to infect its favorite target cell. “They are applying true antiretroviral science to microbicides,” says Mark Mitchnick, who heads R&D for the nonprofit International Partnership for Microbicides in Silver Spring, Maryland.

HIV typically establishes an infection by first attaching to CD4 receptors on white blood cells and then grabbing a second receptor known as CCR5, which normally responds to immune chemicals called chemokines. In the study, clinical immunologist Michael Lederman of Case Western University in Cleveland, Ohio, teamed up with Oliver Hartley of the University of Geneva in Switzerland, whose lab had created a CCR5 inhibitor, PSC-RANTES, by modifying one of the chemokines that uses the receptor. Working with a group led by Ronald Veazey of the Tulane National Primate Research Center in Covington, Louisiana, they applied different doses of the compound to the vaginas of 30 monkeys. Fifteen minutes later, they challenged the animals with an intravaginal dose of a chimeric monkey/human AIDS virus. In animals given relatively high doses of PSC-RANTES, 12 of 15 completely resisted infection. “This is the first paper that says if you target the susceptible cells, you can block infection by mucosal cells,” says Robin Shattuck of St. George's Hospital Medical School in London.

Many mysteries remain about the mechanism of sexual transmission of HIV, and Lederman suggests that this study may help clear up a critical one. Although other studies have shown sexual transmission of the virus through routes that don't involve the CD4/CCR5 nexus, “this experiment suggests that blocking CCR5 is enough to prevent infection,” says Lederman.

Yet he is quick to point out that the dose of PSC-RANTES required for protection in this study is “too high to be practical.” Manufacturing the amount of PSC-RANTES needed to protect each monkey proved extremely expensive, so the Geneva team is now attempting to develop a cheaper version of the molecule. Lederman and others also note that several companies have developed potentially cheaper, small-molecule CCR5 inhibitors. Veazey, working with AIDS immunologist John Moore of Cornell University's Weill Medical College in New York City, last year found that one of these protected two of 11 monkeys in a viral challenge experiment. “We've done better since,” says Moore.

Lederman and colleagues also raise the possibility that their study may have set the bar too high; the monkeys were given hormones to make them more susceptible to the virus. Smaller amounts of PSC-RANTES might therefore work in the real world. Some human studies have shown that the transmission of HIV from male to female may occur as infrequently as one out of every 2000 sexual encounters. But a group led by Christopher Pilcher of the University of North Carolina, Chapel Hill, published a study in the May issue of the Journal of Infectious Diseases reporting that males in the initial stage of an HIV infection can transmit as frequently as once out of every four encounters.

Shattuck says it should be assumed that a microbicide will have to protect against high-dose challenges. Still, he is heartened by the new study. “We've moved from an era of trying unsophisticated approaches to rational drugs that we understand,” Shattuck says. “It's a new phase in microbicide approaches.”

4. CHEMISTRY

# Mass Spectrometrists Salivate Over Recipe for Ions Alfresco

A new way of making ions could revolutionize the venerable practice of mass spectrometry, in which ionized molecules are identified by their weight. Standard ionization techniques work only within cumbersome vacuum chambers or require specially prepared samples. But a simple spritz from a gas jet can liberate ions from almost any surface, even in the presence of air, a team of analytic chemists reports on page 471 of this issue of Science. That means researchers can analyze a vast variety of samples simply by holding them under the jet. The technique could be used in airports to “sniff” luggage for traces of explosives, in orchards to test fruit for pesticide residues, and in many other venues outside the laboratory.

“It's the greatest thing since night baseball,” says John Fenn, a chemist at Virginia Commonwealth University in Richmond. Fenn won a share of the 2002 Nobel Prize in chemistry for developing a technique on which the new method is based. Gary Van Berkel, a mass spectrometrist at Oak Ridge National Laboratory in Tennessee, says the technique has a wealth of potential applications. “My mind's been racing since I read the abstract,” he says. “I came in this morning and set up an experiment, and in 5 minutes I had it working.”

Dubbed desorption electrospray ionization (DESI), the new method combines elements of other well-established techniques, report Zoltán Takáts, R. Graham Cooks, and colleagues at Purdue University in West Lafayette, Indiana. Researchers can ionize large molecules by dissolving them in a solvent and using an intense electric field to pull tiny charged droplets of solution from the end of a needle—an approach known as electrospray ionization, for which Fenn won the Nobel Prize. The new technique uses an electrospray jet differently, to shoot ionized droplets of solvent at a sample.

In that regard, DESI resembles techniques in which beams of other ions or laser light blast ions out of a sample's surface. However, the ion beam technique works only in a vacuum chamber, and laser samples usually must be specially prepared and must fit into the laser rig. DESI works with everyday surfaces and sucks ions into the spectrometer through a sampling tube. Using the method, Takáts and Cooks have detected traces of the explosive RDX on a leather surface and residue of the chemical weapon DMMP on a nitrile glove; tracked organic compounds in seeds and stems of plants; and even sniffed out an antihistamine on the skin of a person who had taken the drug 40 minutes earlier. The team has patented the technique, and a small start-up company will try to commercialize it.

Van Berkel suspects that DESI will prove most useful for analyzing laboratory samples, such as the plates generated in gel electrophoresis measurements. But Albert Heck, a mass spectrometrist at Utrecht University in the Netherlands, says the technique opens the way for taking mass spectrometers out into the world and analyzing surfaces wherever they may be found. As they travel down life's road, mass spectrometrists can now stop and ionize the roses.

# Hughes, NIH Team Up on Novel Training Program

1. Yudhijit Bhattacharjee

The country's biggest private sponsor of biomedical research is joining hands with the National Institutes of Health (NIH) in an unusual arrangement to train interdisciplinary scientists.

Under the initiative, the Howard Hughes Medical Institute (HHMI) will provide up to $1 million over 3 years to each of 10 institutions to help them create Ph.D. programs that integrate biomedicine with the physical sciences and engineering. The money will go toward hiring staff and developing curricula. Once the programs are up and running, the National Institute of Biomedical Imaging and Bioengineering (NIBIB) will provide 5 years of funding to support the actual training of the graduate students. The total cost of the initiative is estimated at$35 million.

The 4-year-old NIBIB already funds training programs at 21 schools around the country. What is unusual about this effort, however, is that HHMI—not NIBIB—will choose the participating institutions. After 3 years Hughes will hand the program over to NIBIB, which will review an institution's progress before providing additional funding. “Although phase II funding is not guaranteed, we expect that all the programs will do well enough to qualify,” says NIBIB's Henry Khachaturian. Each program is expected to train up to 10 students.

HHMI officials approached NIBIB with the idea to “ensure sustainability of the programs that we would be helping to create,” says Peter Bruns, HHMI's vice president for grants and special programs. “It's unrealistic to start a training program without making sure that students will have continued funding.” NIBIB welcomed the opportunity “to foster interdisciplinary training in a planned way,” says institute director Roderic Pettigrew. “HHMI is better equipped than NIH to underwrite and develop the infrastructure for new programs. NIH, on the other hand, is well equipped to support programs that are fully established.”

Although observers like the idea of pooling public and private resources for graduate training, some wonder about the wisdom of having a private foundation, in effect, select grantees for a federally funded program. “If the institutions chosen by HHMI are really the cream of the crop, why do they need a protected competition for funding from NIBIB?” asks one society official, who requested anonymity. A good approach might be “for HHMI and NIBIB to work together on all aspects of selection and administration from day one,” says Peter Katona, director of the Whitaker Foundation, a major supporter of research training in biomedical engineering.

NIBIB officials say the agency will help HHMI select appropriate reviewers and ensure that a majority of them will be available for reviewing phase II applications. Guidelines for the competition, open to any U.S. institution granting Ph.D.s in biology, are online at www.hhmi.org/grants/pdf/comp_annc/2005_nibib_program.pdf.

6. GENETICS

# Disease Backs Cancer Origin Theory

1. David Grimm

Almost all cancer cells have gained or lost entire chromosomes. Despite the genetic turmoil this causes, scientists have disagreed for nearly a century about whether this abnormality and other types of genomic instability, such as that caused by DNA repair defects, are the starting gun for cancer or merely a result of it. A study published online in Nature Genetics this week provides the strongest evidence yet for the starting gun theory by showing that mutations in a gene involved in ensuring proper chromosome number result in childhood cancer.

“The connection between chromosomal instability and cancer is now unassailable,” says Bert Vogelstein, an oncologist at Johns Hopkins University School of Medicine in Baltimore, Maryland. “This study will stimulate a lot of research into whether mutations in genes [involved in chromosome maintenance] contribute to other types of cancer.”

In 1914, German biologist Theodor Boveri noticed that the cancer cells he was studying contained an abnormal number of chromosomes, a state called aneuploidy. The observation led him to postulate that the condition was a root cause of cancer. But as researchers began to discover that mutations in specific oncogenes and tumor-suppressor genes were enough to set cancer in motion, the aneuploidy theory fell out of fashion. Now it's back, thanks to a series of studies in the mid-1990s on the larger issue of genomic instability. For example, Vogelstein and others showed that mutations in genes required for DNA repair led to a hereditary form of colon cancer, indicating that the destabilization of a cell's genome could instigate cancer. But the field is still deeply divided between scientists who believe genomic instability must happen early for cancer and those who say it happens later and may not even be required.

In the new study, a team led by cancer geneticist Nazneen Rahman of the Institute of Cancer Research in Sutton, U.K., screened the DNA of eight families with mosaic variegated aneuploidy (MVA)—a genetic disorder in which more than 25% of a patient's cells are aneuploid and childhood cancers such as rhabdomyosarcoma and leukemia occur much more frequently than normal. In five of these families, the group identified a child with mutations in both copies of a gene called BUB1B. All five children had a high percentage of aneuploid cells, and two have already developed cancer. The gene found mutated in these children encodes a protein previously shown to help guarantee that the right number of chromosomes are passed from cell to cell. The new work is the first to show that defects in BUB1B or any other genes guiding a cell's chromosome partitioning system lead to a human disorder.

“This indicates that aneuploidy has a direct causal role in cancer,” says Rahman. Moreover, she says, the fact that a genomic instability like aneuploidy arises early in the life of someone with MVA argues that it is an incipient event in the disorder's cancer development and not a side effect of other processes. “This study will be a major part of the armory for people who argue that aneuploidy is a cause, not an effect, of cancer,” contends Rahman.

Just because early genomic instability leads to cancer in MVA doesn't mean it's the trigger in all cases, says William Dove, a geneticist at the University of Wisconsin, Madison. His group has been unable to detect this process in a mouse model of intestinal cancer. “Rahman's study provides very important evidence that early aneuploidy can cause cancer,” he says, “but it doesn't close the debate.”

Vogelstein agrees that other cancers should be studied. Unlike the tumors arising from MVA, he says, most cancers are not hereditary. “So it still leaves the door open as to whether this applies to [spontaneous] cancers, … but this is a giant step forward for those who believe early instability predisposes to cancer.”

Establishing an accurate timeline for cancer progression should help researchers develop therapies targeted at preventing and treating the disease. Says Dove, “If we know the nature of the enemy, we will have a better way of attacking it.”

7. ECOLOGY

# Global Survey Documents Puzzling Decline of Amphibians

Almost a third of the world's amphibians are threatened with extinction, according to the first global survey of the situation. And it's not clear what's killing many of them off. “It's very sobering,” comments David Wake of the University of California, Berkeley, about the assessment, described in a paper published online by Science this week (http://www.sciencemag.org%20/cgi/content/abstract/1103538).

Scientists first noticed the perilous state of many amphibians in the late 1980s. Many common species were becoming hard to find, even in national parks and other protected areas. In addition to a loss of habitat, studies pointed to herbicides, stronger ultraviolet light, and a fungal disease called chytridiomycosis. There was also speculation about the role of climate change and invasive species. Despite an accumulating stack of evidence, there was no global picture of all 5743 known species.

The $1.5 million Global Amphibian Assessment project, funded by several federal and nongovernmental donors, was launched in 2001 to provide that global picture. Simon Stuart of the International Union for Conservation of Nature and Natural Resources (IUCN) and colleagues at Conservation International and NatureServe, a biodiversity clearinghouse, began by dividing the world into 34 regions. They assigned a herpetologist to assemble a species list for each region and seek out information such as trends in abundance, distribution, and threats. More than 500 herpetologists reviewed the data. “The effort is unprecedented,” says Michael Lannoo of Ball State University in Muncie, Indiana. The next step was to evaluate the chance that each species would go extinct, according to IUCN “Red List” criteria. Not only are a third threatened, they found, but 7.4% of all amphibians—427 species—qualify for the highest IUCN threat level, known as critically endangered. Moreover, both figures are certainly underestimates, Stuart says, because too little is known about 1294 rare species to gauge their status. Stuart is seeking funding that would allow his team to update the database frequently and review it completely every 3 years. The survey attempted to chart trends in amphibian species as well. One approach was to ask the expert reviewers what was happening to populations. Some 43% of amphibian species are dwindling in numbers, they reported; 27% are stable, and fewer than 1% are increasing. The status of the rest is unknown. Another method was to look at species for which data existed in 1980—when declines apparently began—and compare their Red List status, then and now. The situation has gotten worse over the past 2 decades for 435 species, the survey reveals. (Again, this is likely an underestimate, Stuart cautions, because the decline of many species could have gone undetected.) In North America and Europe, the reason is largely habitat loss, whereas in East Asia it is humans hunting for food. But there is no obvious cause for the declines in the Neotropics and Australia, which host the majority of rapidly declining species. “The bottom line is that there's almost no evidence of recovery and no known techniques for saving mysteriously declining species in the wild,” Stuart says. “It leaves conservation biologists in a quandary.” 8. NOBEL PEACE PRIZE # Kenya's Maathai Wins for Reforestation Work 1. Gretchen Vogel, 2. David Malakoff Arrested, beaten, and jailed for her efforts, environmentalist and political activist Wangari Maathai of Kenya has won the 2004 Nobel Peace Prize. Maathai, 64, is the first African woman to win the prize, announced last week, and the first to be honored for environmental work. The founder of the Green Belt Movement, which since 1976 has organized local groups to plant an estimated 30 million trees across eastern and southern Africa, Maathai was a longtime opponent of Kenya's former strongman Daniel arap Moi. She was physically attacked by opponents on several occasions and was once released from jail only after Amnesty International helped fuel international protests. Since 2002 she has served as deputy environment minister under President Mwai Kibaki and also holds a seat in Kenya's parliament. In awarding the prize, the Norwegian Nobel committee said Maathai “combines science, social commitment, and active politics. More than simply protecting the existing environment, her strategy is to secure and strengthen the very basis for ecologically sustainable development.” Maathai's accomplishment also breaks new ground by recognizing environmental activism as worthy of a prize normally awarded for peacemaking and human-rights advocacy. “Peace depends on our ability to secure our environment,” said Ole Danbolt Mjoes, the Nobel Committee chair. Maathai earned a Ph.D. from the University of Nairobi, one of the first women in the region to do so. She later chaired the school's department of veterinary anatomy, also a first for a woman. Maathai is “delightful, ebullient, and dynamic,” as well as a keen thinker, says Chad Oliver of the Yale School of Forestry and Environmental Studies in New Haven, Connecticut, where Maathai was a visiting scholar in 2002. “She's able to look at a cloud of information and cut right through to the core.” Since winning the award, Maathai has provoked controversy by restating her belief that scientists may have created the HIV virus to harm Africans. Many prominent Africans have endorsed that fringe idea because the epidemic has hit the continent exceptionally hard, says Samuel Kalibala of the International AIDS Vaccine Initiative in Nairobi. But Maathai's remarks are unfortunate, he says: “We should not be diverted from fighting AIDS by trying to blame others.” 9. INFLUENZA: GIRDING FOR DISASTER # Looking the Pandemic in the Eye 1. Martin Enserink Researchers have no way of knowing what the next influenza pandemic will look like. But models and educated guesses are disconcerting Ask flu experts about their worst nightmare and they may tell you something like this. Somewhere in Asia, a new flu virus is born that's able to jump from one human to the next, yet is cloaked in avian proteins that human immune systems have never seen before. Laying low at first, the virus sickens and kills a small number of people, while it's getting better at the human-to-human transmission game. When authorities finally notice the expanding cluster of flu cases, the virus has already moved on. It takes advantage of flights that connect Asia's major cities to the rest of the world, popping up simultaneously in Sydney, Los Angeles, and London. Hundreds begin to die, literally drowning as fluid fills their lungs. A stunned public demands a vaccine, drugs—anything—but no vaccine will be available for months, and antivirals are in short supply; the question is, who gets them? Panic and riots erupt while schools, businesses, and transportation systems are shutting down. Overcrowded hospitals start turning away desperate patients. There aren't nearly enough doctors and nurses to take care of the sick and dying, nor enough coffins. When the outbreak finally peters out 18 months later, more than 2 billion people have become ill, and more than 40 million are dead—twice the number claimed by AIDS in 25 years. True, that's a worst-case scenario—but few experts dismiss it out of hand. After years of neglect, the threat of a new pandemic is back on the world's radar screen, beeping noisily. Public health experts, virologists, and disease modelers are struggling to envisage how fast it would spread, how many it would kill, what it would cost, and most of all, how best to fight it. The efforts were spurred in part by severe acute respiratory syndrome (SARS), the planet's close brush with pandemic disaster last year. The SARS virus wasn't all that contagious, striking fewer than 9000 people before it was brought under control. But the world may not be so lucky next time. Nor does it take a newcomer like the SARS virus for a pandemic to occur. Most experts agree that flu strains now circulating can, and eventually will, spawn a new pandemic. Predicting what it will look like means going out on a limb, however, because everything depends on which flu strain is the culprit and how virulent it is—two questions no one can answer. Still, researchers can crunch the numbers for a range of assumptions. They end up with a series of scenarios—from something quite benign to an “overwhelming and potentially catastrophic event,” says Martin Meltzer, an economist and disease modeler at the U.S. Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia. Even trickier to predict are a pandemic's social, political, and economic fallout. “Go ask the fiction writers what could happen,” Meltzer says. It seems certain, though, that a pandemic will raise agonizing dilemmas about who should be first to receive drugs, vaccines, and medical care—an issue that most countries haven't even begun to debate. ## Virgin territory Flu pandemics occur when a new virus emerges that's easily transmissible between people and also finds virgin territory in the human population because no one is immune. This happens when one or both of the virus's envelope proteins (hemagglutinin and neuraminidase, the H and N in names like H5N1) have never before circulated in humans. By far the most terrifying example is the 1918–19 “Spanish flu” pandemic, during which at least 20 million people, and perhaps as many as 100 million, are believed to have perished. Most of that virus's genetic baggage has been reassembled from preserved tissue scraps and an Alaska victim's frozen body. In a paper published in last week's issue of Nature, researchers reported that a modern flu strain equipped with the 1918 hemagglutinin is highly pathogenic to mice—a finding that may help clarify why the 1918 virus was so deadly. It's still unclear where the virus came from, however; nor are researchers sure about the origins of two subsequent, milder pandemics that struck in 1957 and 1968. For decades, the dominant theory was that new pandemic viruses arise when avian and human flu viruses reassort, or hybridize, inside pigs, which can be infected with both. (Chinese farms, where ducks, humans, and pigs mingle, were seen as plausible locales.) But since 1997, three avian flu viruses—including H5N1, the virus that has infected poultry in 10 Asian countries—have been found to infect humans directly. Now, the predominant worry is that humans infected with both avian and human viruses may be mixing vessels. Fortunately, chances of this happening still seem low, says Neil Ferguson, an epidemiologist at Imperial College in London. Even if you assume that reassortment occurs in each and every patient infected with the two viruses—which is unlikely—more than 600 people would have to be infected with H5N1 to create a 50% chance of reassortment, Ferguson and his colleagues wrote earlier this year in Science (14 May, p. 968). So far, fewer than 50 people in Vietnam and Thailand are confirmed to have been infected with H5N1. What's more, most reassortants are likely to pose no threat. Assuming a new pandemic virus emerges, how might it behave? Epidemics can be modeled several ways, but mathematicians always need a number of key parameters, such as the basic reproductive number (R0), which denotes the number of secondary infections resulting from one patient, the attack rate (the percentage of people who get sick after being exposed to the virus), the chance of becoming infected when in close contact with a patient, the incubation period, and the mortality rate. For many diseases, those variables are reasonably well known and more or less constant. Not pandemic flu; even year-to-year changes in the influenza virus make for difficult modeling, says Ira Longini, an expert at Emory University in Atlanta—which is why modelers have tended to stay away from flu. But faced with what many perceive as a gathering threat and using past pandemics as a rough guide, modelers are beginning to tackle the problem. The Models of Infectious Disease Agent Study (MIDAS), for instance, a network funded by the U.S. National Institutes of Health that includes Longini's group, this summer made work on flu pandemics its top priority. The U.S. government is keenly interested in the results, Longini says, because models can help decide how best to deploy drugs and vaccines. The models all suggest that pandemic flu is unlikely to be contained using the old-fashioned public health measures that put the SARS genie back into the bottle, such as isolating patients and tracing and quarantining contacts. SARS has an incubation period of about 6 days during which infected people don't seem to infect others—precious time health authorities could use to trace those exposed but still healthy. With flu, they'd have only about 2 days on average. Moreover, SARS's severe symptoms helped identify patients, whereas flu can be as mild as the sniffles. The only exception may be very early on, notes Ferguson. When the virus is still struggling to replicate among humans, surveillance and quarantine, perhaps helped by aggressive use of antiviral drugs, might nip a pandemic in the bud—which is why the World Health Organization is exploring a plan to ship antivirals to the cradle of a potential pandemic (see p. 394). Once a virus was on the loose, jumbo jets would likely spread pandemic flu faster than ever in history. In a model published last year, Rebecca Grais and her colleagues at Johns Hopkins University in Baltimore, Maryland, collected data on the number of passengers traveling daily among 52 major cities around the globe and then calculated how fast the 1968 strain would have spread had it surfaced in 2000. Although the model has its limits, the trend is clear: The outbreak would peak in most of the 52 cities within 6 months (see graphic above). In the same model fed with travel data from 1968—as well as in the actual pandemic—almost a year passed before the virus made it around the globe. The difference is crucial, because developing and mass-producing a vaccine may take as long as 6 months. Few countries can hope to be spared that long. ## Two waves The toll of the pandemic would depend largely on the attack rate and the mortality rate—two unpredictable factors that can change during an outbreak. Spanish flu, for instance, came in two waves: One, in the spring and summer of 1918, caused widespread disease but few deaths; another, much more vicious wave the following autumn and winter killed half a million people in the United States alone. Presumably, the virus had evolved to become more virulent. When trying to predict the course of the next pandemic, however, most modelers look more to 1957 and 1968 than to 1918. That's in part because much more is known about the virology and epidemiology of those epidemics, which makes modeling easier. Still, Longini admits that the later pandemics make for rosier outlooks, and the MIDAS group is now collecting data to tackle the 1918 pandemic. When Meltzer and two CDC colleagues estimated the economic impact of a pandemic on the United States in a 1999 study, they used conservative attack and mortality rates comparable to those in the milder pandemics. Even then, a pandemic could cause between 314,000 and 734,000 hospitalizations and claim between 89,000 and 207,000 lives, they found. Even the lower figures would overwhelm the U.S. health system, says Meltzer: Hospitals were under severe stress when the 1999–2000 flu season was worse than usual. The team put the economic cost of a 1968-style pandemic for the United States at somewhere between$71.3 billion and $166.5 billion. Using a different set of assumptions, including lower health care costs, Jeroen Medema of Solvay, a vaccine company in the Netherlands, arrived at about$167 billion for all developed countries combined. Both studies, however, included only direct medical costs and lost productivity as a result of disease and death. A pandemic would almost certainly cause economic disruption that would multiply the cost several-fold. (Asian economies suffered incalculable losses from the SARS outbreak.)

Vaccines would curb the toll, but supplies would be short in the beginning, Meltzer says—as would drugs and attention from doctors and nurses. “Who will get a hospital bed—a 90-year-old grandmother or a 30-year-old mother of two children? People in America are not used to that kind of rationing,” Meltzer says, although they're getting a taste of it now that manufacturing problems have abruptly cut the yearly flu vaccine supply in half (see p. 385).

In an as-yet-unpublished paper, Longini and his colleagues show that, when a vaccine is in short supply, different objectives can lead to radically different strategies during relatively mild pandemics. When reducing mortality is the primary goal, for instance, it's best to vaccinate the elderly. When trying to reduce the number of cases or reduce the economic fallout, it would be better to start with schoolchildren.

But so far, there's been little discussion about such priorities and even less consensus. When CDC and other organizations convened a meeting of more than 125 public health experts from 46 states in 2002, participants were asked which of five goals should get top priority during a pandemic: reduce disease, reduce deaths, ensure that essential services continue, limit the economic impact, or ensure “equitable” distribution of scarce resources. None received more than 50% of the votes. “We need a national debate now about these questions,” Meltzer says. “When you have a pandemic, it's not a good time to have a discussion with your doctor about the ethics of rationing.”

If handled badly, such choices may increase the risk of social upheaval, says Monica Schoch-Spana, a senior fellow at the University of Pittsburgh's Center for Biosecurity. Today's public is likely to become disillusioned when it finds that the government can't offer protection. “There's always the operating assumption that some expert somewhere knows what to do,” she says. Clearly explaining the choices as well as the uncertainties is going to be essential, she says.

Retired historian Alfred Crosby, an expert on the 1918 pandemic, is worried about panic, too. But it needn't happen, he notes—the next pandemic may be of the mild rather than cataclysmic variety. Says Crosby: “I wish us all luck.”

10. INFLUENZA: GIRDING FOR DISASTER

# Facing Down Pandemic Flu, the World's Defenses Are Weak

1. Jocelyn Kaiser

A lack of interest in developing pandemic flu vaccines and a dearth of antiviral drugs have left the world vulnerable to a global outbreak

At a hotel meeting room outside Quebec last March, 35 health officials and others from the world's seven leading industrialized countries and Mexico passed around a vial of bitter-tasting white power. If Asia's potent H5N1 bird flu assumes a form transmitted between humans, this drug, oseltamivir, would be the world's only initial defense against a pandemic that could kill millions of people. But oseltamivir, sold as Tamiflu, is made by only one company, Roche, at a single plant in Switzerland. “We are living in a brave new world where we only have one drug,” says flu expert Arnold Monto of the University of Michigan, Ann Arbor, who spoke before the working group meeting of the G7+ Global Health Security Action Group.

That grim assessment is one indicator of the world's vulnerability to pandemic influenza. Most virologists say a pandemic is a virtual certainty within the next few decades, if not from H5N1 then from another avian flu strain (see p. 392). When that happens, public health officials will have two tools to battle the disease: antiviral drugs and vaccines. But although research has produced effective new antivirals, they are expensive, and global supply falls far short of need. And a promising genetically engineered vaccine against H5N1 is still an experimental product only just now being tested in people.

After years of warning from flu experts, governments are finally beginning to respond. Some countries are starting to stockpile antivirals. The United States in August unveiled a draft pandemic flu plan; it is also launching clinical trials of an H5N1 vaccine and will pay Aventis Pasteur $13 million to manufacture 2 million doses. “There's a lot of momentum,” says virologist Robert Webster of St. Jude Children's Research Hospital in Memphis, Tennessee. But even that is not enough, say global flu experts. Of the world's 12 major flu vaccine manufacturers, so far only two are willing to tackle the financial, regulatory, and patent issues involved in making a new pandemic vaccine, mainly for the U.S. market. Companies in other countries also need to be developing emergency products, flu experts say. Moreover, only 15 countries have pandemic flu preparedness plans that lay out how scarce vaccines and antivirals will be distributed, notes World Health Organization (WHO) virologist Klaus Stöhr. As worries intensify, flu experts are exploring a controversial alternative: pooling available supplies of antiviral drugs to stamp out an incipient pandemic in Asia. But whether countries will voluntarily ship their own precious stockpile overseas to fight a faraway plague remains to be seen. ## A clear and present danger The United States last geared up for pandemic flu in 1976, after swine flu broke out in Fort Dix, New Jersey. Within 10 months, the country produced 150 million doses of vaccine and vaccinated 45 million people. But the virus didn't spread, and critics said the government had jumped the gun. That led to the first U.S. pandemic flu plan. The need to rethink such plans became apparent in 1997, when an outbreak of H5N1 avian flu in Hong Kong killed six people. Unlike previous pandemic strains, H5N1 did not first combine with a human flu virus in pigs; instead it jumped directly to infect humans. This transmissibility and the virus's potency raised the risk that the avian virus could mix with a human flu virus inside a person to yield a deadly pandemic strain. Worries intensified when researchers realized that the tried-and-true method for making flu vaccine in eggs probably would not work with the new avian strain. Flu vaccines are traditionally made by infecting eggs with a target virus and a nonpathogenic strain that grows well. In the eggs the viruses mix their eight genes. Manufacturers then select a strain with genes for neuraminidase and hemagglutinin (two glycoproteins on the virus's surface) from the target virus, and the rest from the normal flu strain; inactivated virus is then used to make vaccine. But H5N1 kills eggs. A solution exists: reverse genetics (Science, 27 February, p. 1280). Using this technique, the two genes for neuraminidase and hemagglutinin, as well as the six genes from a safe virus, are cloned in bacterial DNA and then reassembled. With highly virulent strains like H5N1, the hemagglutinin gene is first modified to reduce its pathogenicity so the seed virus can be grown in large quantities in eggs. Using reverse genetics, teams at St. Jude and the U.K.'s National Institute for Biological Standards and Control (NIBSC) each produced an attenuated Vietnam H5N1 strain within 3 to 4 weeks earlier this year—“clearly a phenomenal advance,” notes Iain Stephenson of the U.K.'s Leicester Royal Infirmary. Making a candidate vaccine is just the first step; it then has to be tested in humans. Trials of pandemic-like vaccines in the 1970s and since have found that because people have no previous exposure to these viruses' coat proteins, they will likely need two doses plus high levels of antigen. Even then, the vaccine may not work without an adjuvant, a compound that makes the vaccine more immunogenic. To assess dosage for the reverse-genetics vaccine against Vietnam H5N1, the U.S. National Institute of Allergy and Infectious Diseases (NIAID) expects to begin clinical trials later this year, using lots made by Chiron and Aventis Pasteur from the St. Jude seed strain. Last month, the U.S. Department of Health and Human Services (HHS) also announced that Aventis Pasteur will manufacture antigen for perhaps 2 million doses, depending on how much the clinical trials show is needed. Besides providing a stockpile for health workers exposed to H5N1, “we want to get these manufacturers playing with it” so they can design adequate worker protections and see if the vaccine grows well in eggs, says NIAID's Linda Lambert. The institute also plans to test a vaccine against H9N2, another bird flu strain. As part of the U.S. draft pandemic flu plan, HHS also disbursed$50 million this year and plans to spend $100 million in 2005 to help ensure that companies have enough eggs year-round. The funds will also support development of an alternative to using eggs—producing vaccine with cell culture using fermenters—an advance that should eventually expand “surge capacity.” Under the U.S. plan, “potentially everybody” would get pandemic vaccine, says Bruce Gellin of HHS, although no timeline has been set for reaching this goal. ## Supply-side economics So far, only the United States is putting serious money into testing reverse-genetics flu vaccines. And the country is operating “with its own interests in mind,” says Stöhr—not to supply the world. (Outside the United States, Japan has plans for trials starting next year, and Aventis Pasteur in France is making test lots of NIBSC's H5N1 seed strain for European trials.) David Fedson, a retired former medical director for Aventis Pasteur in France, points out that companies in just nine countries in Europe produce 85% of the world's flu vaccine, so if governments decide to impound vaccine to protect their populations (as the United States did during the 1976 swine flu episode), other countries will be in trouble. The United States—which has only one major domestic supplier, Aventis Pasteur in Swiftwater, Pennsylvania—is getting a preview of this scenario this fall, after possible contamination at Chiron's U.K. facility halted use of about 47 million doses of vaccine, half the supply destined for the United States (see p. 385). Moreover, the world's capacity for making a monovalent pandemic flu vaccine is now 900 million doses, enough for only 15% of the world's population. To stretch the supply, researchers will almost certainly need to use an adjuvant—one that's both cheap and plentiful. Some experts are buzzing about a small trial by GlaxoSmithKline researchers who found that if they used alum to boost an H2N2 vaccine, they needed only 1.875 micrograms of antigen, 12.5% of the normal dose. Alum would also be cheaper than MF59, the adjuvant NIAID plans to test. Adding alum could potentially allow companies to vaccinate 3.5 billion people, or half the world, with two doses of H5N1 vaccine, Fedson says. NIAID isn't pursuing this strategy, however, because no flu vaccine with alum adjuvant has been licensed in United States. “This is a concern,” agrees NIBSC's John Wood. WHO's Stöhr has urged European Commission (EC) leaders to take the initiative in contracting with companies in Europe to test a low-dose pandemic H5N1 vaccine containing alum adjuvant. However, the commission has not yet found the money. “The EC has not the flexibility or the political will,” Stöhr says. Companies have little incentive to test pandemic vaccines for a market that may never materialize. Intellectual-property and liability issues are also major deterrents. The reverse-genetics flu vaccine is licensed by MedImmune, which uses technology from St. Jude. But Mount Sinai School of Medicine and the University of Wisconsin have patents on similar technology. MedImmune has licensed it for research purposes to Aventis Pasteur and Chiron, but if these companies or others wanted to market a vaccine, they would need an agreement with the other patent holders, says Hugh Penfold of the Centre for the Management of IP in Health R&D, a nonprofit in Oxford, U.K. (The U.S. government can assert its patent rights to produce domestic vaccine, but it could not be sold abroad.) Because a reverse-genetics vaccine is considered a genetically modified organism, it would also need special clearance in Europe. However the vaccine is made, countries would need to pass legislation to shield companies from liability should the vaccine cause serious side effects, as did the swine flu vaccine. Some believe these problems will quickly be solved if a pandemic arrives. “What happens in a crisis is, a lot of the roadblocks get moved,” says virologist Maria Zambon of the U.K.'s Health Protection Agency. Meanwhile, Stöhr notes, countries can get a head start by boosting their capacity to make and deliver regular flu vaccine. Ontario, Canada is a model: Since 2000, the province has offered everyone a free regular flu shot. (Earlier this year, Canada also unveiled a pandemic plan that includes paying one company to manufacture pandemic vaccine for all 32 million Canadians.) Fedson notes that a similar policy in the United States could help guarantee annual flu vaccine supplies and avoid debacles like this year's vaccine shortage, which he hopes will be a “watershed event.” ## Stopgap measures Even if companies worldwide had the ability and commitment, it could still take 4 to 6 months to manufacture a reverse-genetics vaccine matching a new pandemic flu strain. That leaves antivirals as the first response. Of the two classes of flu antivirals, those that, like Tamiflu, target neuraminidase are considered the best choice because the flu virus is less likely to develop resistance. Roche says that preclinical studies suggest that Tamiflu will be effective against H5N1. Ira Longini, a modeler at Emory University in Atlanta, estimated in a 1 April 2004 paper in the American Journal of Epidemiology that a course of antivirals given prophylactically to 80% of the exposed U.S. population for 8 weeks could be as effective as a vaccine in preventing deaths and disease. Although that could require up to 2 billion doses, an unrealistic number, less would be needed if the virus appeared only in some locations. Some countries, such as Australia, are building sizable stockpiles of Tamiflu. Japan has enough for 20% of its population; the United States can treat 1 million people and hopes to acquire more of the drug. But not all countries can afford Tamiflu, which costs$8 to $10 per course (two pills a day for 5 days) in bulk, Monto notes. And Roche can only make 7 million treatments a year right now (although the company says it can meet all current orders and is expanding capacity). Most developing countries are in far worse shape. A meeting organized by the Sabin Vaccine Institute in New Canaan, Connecticut, later this month will explore ways to increase vaccine manufacturing capacity in countries such as India. But Africa is “a big, big, question. Without a doubt, the virus will get there. … The situation will be much, much worse than anywhere else. Access to vaccines will not be an option, let alone antivirals,” Stöhr says. With preparations lacking, some experts are mulling whether a mobile stockpile of antivirals could be used to wipe out an incipient pandemic at the source by treating everyone in contact with a patient. This might be feasible, given improvements in WHO surveillance for potential pandemic flu viruses, says Nancy Cox of the U.S. Centers for Disease Control and Prevention. HHS is spending$5.5 million to help countries in Asia begin or improve surveillance for human flu strains, she adds.

Some experts suspect that a pandemic hybrid virus will not be very efficient at human-to-human transmission at first, so it will spread slowly. “We might have a narrow window of opportunity to extinguish it before it becomes a wildfire,” says Stöhr.

A consortium of modelers funded by the U.S. National Institutes of Health, including Longini, is looking at the feasibility of stopping a pandemic in Asia if, say, 1 million or 2 million courses of antivirals were available, Cox says. They will present preliminary results at a meeting at Emory in late October. But even if the models suggest it would work, rich countries would need to agree to share their drugs, Stöhr says. The question may be whether an agreement can be reached before the next pandemic arrives.

11. INFLUENZA: GIRDING FOR DISASTER

# Searching for All-Powerful Flu Weapons

1. Jocelyn Kaiser

Influenza virus is a shape-shifter, constantly mutating into new pathogenic strains. Every year, companies have to design an entirely new flu vaccine to match the predicted strain's outer coat of proteins. Likewise, to fight a new pandemic strain, researchers would have to start from scratch (see main text), a process that could take 6 months. The Holy Grail of flu research is a vaccine that works against all strains. Many labs and companies are working on this, as well as more effective antiviral drugs. Possible approaches include:

DNA vaccines. To create a universally protective flu vaccine, researchers are focusing on virus proteins that are conserved among strains or that don't mutate much. A team led by immunologist Suzanne Epstein of the U.S. Food and Drug Administration has shown that a DNA vaccine containing genes for an inner protein, NP, as well as M (matrix) proteins, can work against avian flu. These vaccines deliver strands of DNA into cells, causing the cells to make the antigen themselves. This stimulates various immune responses, including T cells, that provide broader immunity than do vaccines containing only antigen. Live virus also does this, but DNA vaccines are safer and can be produced quickly.

As Epstein's team reported in Emerging Infectious Diseases in August 2002, their vaccine, injected into mice, provided partial protection against two strains of H5N1 avian flu. The mice still got sick from the more virulent strain, but half survived a challenge dose that otherwise would have killed them. Such a vaccine could be used to reduce mortality until a matching vaccine became available, Epstein suggests. Others are working on ways to get DNA vaccines to provoke an even stronger immune response, for example by boosting gene expression, using bioengineered proteins, or including additives called adjuvants.

New antiviral drugs. To improve on traditional antivirals, molecular biologist Robert Krug's lab at the University of Texas, Austin, is targeting a flu virus protein called NS1 that shuts down the cell's own production of virus-fighting proteins. Because the virus can't avoid using NS1, “we know this is an excellent target,” Krug says. A collaborating lab has begun screening for molecules that block NS1 and could be potential drugs.

The problem may be getting companies interested, Krug says. He points to the fate of Relenza (zanamivir), an inhaled drug that may be more impervious to flu virus resistance than oseltamivir, or Tamiflu, the leading flu drug. GlaxoSmithKline cut back its marketing of Relenza in 2000 in response to disappointing sales

12. INFLUENZA: GIRDING FOR DISASTER

# Vaccinating Birds May Help to Curtail Virus's Spread

1. Dennis Normile*
1. With reporting by Xiong Lei in Beijing.

As avian influenza continues to ravage Asian poultry, countries are experimenting with a novel control strategy

Fearful that a deadly flu epidemic could be brewing in Asia, some countries are stockpiling drugs, preparing pandemic flu plans, and ratcheting up vaccine production (see p. 394). As these efforts kick into overdrive, animal experts are grappling with the other half of the bird flu equation: the birds. Specifically, they are debating whether a relatively untested strategy of mass vaccination of chickens and other poultry against avian flu will do more harm than good in warding off a human pandemic.

Since its appearance in 1997, global health experts have worried that H5N1 will combine, or reassort, with a human flu virus to produce an easily transmissible strain with H5N1's lethality. To avert such a disaster, last winter and spring seven Asian countries slaughtered more than 100 million birds, decimating the poultry industry. But the virus has resurfaced and appears to be endemic in the region. And the more virus in circulation, the greater the chance of a deadly reassortment.

Animal health officials agree that the best ways to curtail H5N1 are increasing surveillance and improving biosecurity, which includes a host of measures intended to prevent diseases from spreading among flocks and to the public. But now, after years of debate, consensus is building that vaccination of at-risk poultry could also be a critical tool in averting a human pandemic. Indeed, in September, alarmed at the spread of H5N1, the Paris-based World Organization for Animal Health (OIE) and the United Nations Food and Agriculture Organization (FAO) strengthened a previous recommendation encouraging consideration of vaccination in conjunction with other control methods.

But there's a catch, explains Alex Thiermann, a veterinarian at OIE: “If improperly done, vaccination could be dangerous.” It could enable the virus to circulate undetected among birds, perhaps spurring its evolution. And no matter how helpful poultry vaccination might be, some countries may decide against it for fear that it would jeopardize their export market.

So far, Hong Kong requires vaccination of all poultry. Thailand forbids it. China and Indonesia are selectively vaccinating in regions where the virus has appeared.

## Risks and benefits

The clear benefit of vaccination is its ability to reduce the amount of wild virus in circulation. Although vaccination does not always prevent infection—just disease—it takes a much higher dose of virus to cause infection, and vaccinated birds that do become infected shed far less virus than unvaccinated birds. As an added precaution, animal health experts agree that vaccinated birds that become infected should be culled. “By reducing the amount of virus in the environment, you reduce the possibility of the virus spreading to a new flock, and you reduce the risk to humans,” says David Suarez of the U.S. Department of Agriculture's (USDA's) Southeast Poultry Research Laboratory in Athens, Georgia.

For a country to undertake vaccination safely, it first must ensure the quality and efficacy of the vaccine. It must be targeted to the virus in circulation, properly inactivated, and tested to determine the adequate dosage.

Then there's the problem of distinguishing vaccinated birds from birds infected by the wild virus. If the vaccine is derived from the circulating virus, both infected and vaccinated birds would appear positive in antibody tests. This problem has limited the use of avian flu vaccines in the past because it prevents epidemiologists from tracking the circulating virus. It could also make it hard to prove that flocks are disease-free so exports can resume once the disease is stamped out. (The use of vaccines to control highly pathogenic avian influenza is so new that there are few precedents to follow in resuming trade once an outbreak is contained.)

Long-term experience with an avian vaccine in Mexico has raised other concerns, as reported by Suarez and colleagues in the Journal of Virology in August. Farmers in Mexico have been immunizing chickens against a low-pathogenicity H5N2 virus with the same vaccine for 7 years. Over time, the virus has mutated, in a process called antigenic drift. Although the vaccine still prevents clinical disease, it no longer reduces the amount of virus shed by the chickens. Suarez believes that widespread vaccination probably contributed to the virus becoming endemic not only in Mexico but in neighboring Guatemala and El Salvador as well. To avoid this, the virus must be monitored and the vaccine updated periodically.

## A shift in favor

Despite these hurdles, sentiment began to shift in favor of adding vaccination to other avian flu control measures several years ago. With the increased scale of modern poultry farms, culling in a buffer zone around an infected flock was killing enormous numbers of healthy birds. Some farmers and animal health officials began arguing that vaccination in a buffer zone, instead of slaughter, might be more humane and cost effective.

In addition, studies done at the USDA lab in Georgia and reported in Avian Pathology in 1999 and in Vaccine in 2000 showed that a vaccine based on one H5 virus subtype might provide cross-protection against several others. If so, vaccinating with a strain that differs from the circulating strain could solve the problem of differentiating vaccinated-but-uninfected birds from infected birds. More recently, researchers at the Tai Lung Veterinary Laboratory of Hong Kong's Agriculture, Fisheries, and Conservation Department tested a vaccine based on an H5N2 strain against the H5N1 strains that caused outbreaks in Hong Kong in 1997 and 2002. Trevor Ellis, senior veterinary officer at the Tai Lung lab, says the vaccine “protected against clinical disease and produced greater than 1000-fold reduction in virus excretion in birds given heavy virus challenge doses.”

More convincing than the lab studies was Hong Kong's experience. Since H5N1 first surfaced there in 1997, the territory has progressively strengthened H5N1 biosecurity measures. Despite these efforts, Hong Kong has repeatedly been hit by H5N1 outbreaks. During an outbreak in December 2002 and January 2003, a number of farms were infected. On three of these farms, chickens in infected sheds were culled, but chickens in other sheds were inoculated with a vaccine based on the H5N2 strain. The virus spread to additional sheds on two of these farms, killing some of the recently vaccinated chickens. But as Ellis and his colleagues reported in the August issue of Avian Pathology, 18 days after vaccination, when immunity had developed, there were no new cases of disease among the vaccinated birds; intensive monitoring found no evidence of asymptomatic shedding.

In early 2003, Hong Kong added universal vaccination to its control measures. Unvaccinated “sentinel” chickens are placed within each flock, and there is regular serologic and virologic testing. When H5N1 swept through neighboring China early this year, Hong Kong remained virus-free.

Last winter, both South Korea and Japan identified H5N1 outbreaks quickly enough to contain them with limited culling, still the preferred approach. But where stamping out is impractical or uneconomical, vaccination should be considered, says Joseph Domenech, chief of animal health services for FAO.

Hong Kong's experience is not easily translated to other countries, however. Hong Kong's poultry industry is limited to just 150 farms and a handful of families raising backyard chickens. The territory is small and has an infrastructure capable of fully monitoring the use of vaccines. Hans Wagner, FAO's regional director, says, “It's a substantial challenge to extend these measures to an entire country”—and expensive. The vaccine alone costs about 7 cents per bird, not counting the labor of injecting or the monitoring that should accompany it. By contrast, FAO consultants and others who have visited China and Indonesia—which are both vaccinating in areas where H5N1 has been reported—noted several shortcomings. Several of the vaccines in use in both countries are based on the H5N1 strain itself, making it difficult to track the disease. And the use of unvaccinated sentinels and the serological and virological monitoring is spotty at best.

In Thailand, which has reported more than 250 outbreaks in 45 of the country's 76 provinces in the last 3 months, authorities have rejected vaccination, at least for the moment. Yukol Limlamthong, director-general of Thailand's Department of Livestock Development, says they are worried that vaccination might enable the virus to circulate silently among vaccinated birds, exposing farm hands and families to infection. “We don't want to put them at risk,” he says. But flu experts elsewhere suspect that commercial concerns factored heavily in the decision.

The OIE Terrestrial Animal Health Code, which governs international trade in animals and animal products, says a country can be considered free of avian influenza if specified levels of surveillance do not turn up the virus—regardless of whether it is vaccinating. But the code is vague and places the burden of proof on the exporting country. Johan Reyniers, a press spokesperson for the European Commission in Brussels, says, “It would ultimately be up to Thai authorities to demonstrate that vaccination is properly implemented.”

For now, Thai officials believe it will be easier to convince trading partners that its poultry products are safe if the country can control the disease without vaccination. But whether it can remains to be seen.

13. INFLUENZA: GIRDING FOR DISASTER

# Asia Struggles to Keep Humans and Chickens Apart

1. Dennis Normile

SONG PHINONG, SUPHANBURI PROVINCE, THAILAND—After having 30,000 chickens culled when H5N1 turned up on a farm 2 kilometers away, Boonchu Taeng-orn got serious about biosecurity. When permitted to restock his farm here in the central lowlands 2 hours north of Bangkok, he followed recommendations of Thailand's Department of Livestock Development to the letter. He strung netting from the shed roofs to the tilapia ponds beneath to keep wild birds out. (Biosecurity experts discourage locating chicken coops near open water, but raising tilapia on bird droppings is key to the economics of chicken farming here.) As few workers as necessary go into the sheds, changing first into work clothes kept at the site, walking through a disinfecting mist, and stepping in pails of disinfectant on the way in. The egg crates are disinfected before use, as are vehicles at the gates to each compound. And Taeng-orn follows the all-in, all-out practice: When he fills a shed with new chicks, he keeps them until egg production drops and then sells the entire batch. Sheds and cages are washed and repaired before the next batch arrives. “The emphasis on cleanliness is definitely good. It is more humane for the animals and safer for the workers,” Taeng-orn says.

It is also safer for the world. Infectious disease experts agree that keeping zoonotic diseases like H5N1 and severe acute respiratory syndrome from crossing the species barrier into humans will partly depend on the efforts of millions of farmers like Taeng-orn. A greater challenge is to extend such practices to the numerous households that keep backyard chickens. Alex Thiermann, an official with the World Organization for Animal Health, says that large poultry operations in Asia have biosecurity practices on par with farms in the United States or Europe. But in the backyards, there is “no biosecurity at all.”

A key element of Thailand's push to stamp out H5N1 is to educate small holders and require that even backyard chickens be kept in coops to minimize contact with wild birds and family members. Vietnam, too, has launched an education campaign targeting small chicken operations. But no one expects sudden changes in such an age-old practice.

Hong Kong is taking aim at another entrenched custom: It is considering closing its live animal markets. Currently, buyers pick a live chicken at one of more than 800 live animal shops and have it slaughtered on the spot. K. Y. Yuen, a microbiologist at the University of Hong Kong, favors a central slaughtering facility, both to reduce the chances of exposing the general public to avian influenza and to cut the incidence of other infections. “Other advanced countries adopted central slaughter long ago,” he says. The government asked for public comment this summer and is now deciding how to proceed.

14. NOBEL PRIZES

# Laurels to Three Who Tamed Equations of Quark Theory

1. Charles Seife

It might be fun to blow things up, but this year's winners of the Nobel Prize in physics earned the plaudits of their colleagues with a discovery that does the opposite: It prevents equations that describe one of the fundamental forces of nature from exploding.

The three new laureates, Frank Wilczek, David Gross, and H. David Politzer, discovered a property of the strong force—the force that glues quarks to one another—known as “asymptotic freedom.” Not only did the idea explain some baffling experimental results in particle colliders, but it also showed how to keep the equations that describe the strong force from producing troublesome infinities. “They made the discovery and saw the significance of it,” says Niels Kjaer Nielsen, a physicist at the University of Southern Denmark in Odense. “[The prize] is fully deserved.”

Particle physics is swimming with infinities: places where the equations that describe the behavior of a particle seem to blow up into a meaningless jumble of singularities. One reason is that every region of space, even the deepest vacuum, is seething with “virtual” particles that pop in and out of existence—and these particles make even the simplest concepts very difficult.

For example, an electron is surrounded by a cloud of evanescent particles. When scientists try to gauge its charge, the cloud “screens” the naked electron and hides some of the charge from view. If you could somehow worm a measuring instrument through the cloud, getting closer and closer to the bare electron at the center, you would see the measured charge get greater and greater as you penetrate the screen of virtual particles. Strictly speaking, the plain-vanilla equations of the Standard Model of particle physics say that the charge increases without bound at smaller and smaller distances. In other words, the equations blow up. Scientists have come up with mathematical coping mechanisms to get around this problem; the 1965 and 1999 physics Nobels were given for figuring out how to deal with these sorts of infinities in different contexts.

In the early 1970s, physicists studying the strong force were beating their heads against a similar problem. But the infinity-coping techniques developed for the electric force (and for the weak force, which is responsible for phenomena such as nuclear decay) didn't work for the strong force— until Wilczek, Gross, and Politzer made a counterintuitive discovery.

In 1973, Politzer, currently at the California Institute of Technology in Pasadena, and, separately, Wilczek, at the Massachusetts Institute of Technology, with Gross, at the Kavli Institute for Theoretical Physics in Santa Barbara, California, realized that, unlike the electric (and weak) forces, the strong force gets weaker at close range—much as a taut spring relaxes when the ends are brought close together. As a result, virtual particles “screen” quarks in a very different way from how they screen electrons: The virtual particles—gluons—that surround and interact with a quark feel one another's presence in a way that the virtual particles that surround and interact with an electron—photons—don't. Stick a particle right next to a quark, and it wouldn't feel the strong force at all; it would be “asymptotically free” from the strong force, and quarks forced into close proximity would behave more or less like hard little unbound particles rather than a sticky clump. That is precisely what experimentalists at the Stanford Linear Accelerator Center in California had found a few years earlier by scattering electrons off protons. Turned around, asymptotic freedom explains why quarks are never found roaming free from one another: At large distances and low energies, the strong force is too powerful to overcome.

Particle theorists have long anticipated this award, and Wilczek was no exception. “I'd be lying if I said it was unexpected,” he said with a laugh. “I thought it was an important theory, and the data in favor of it has been clear for at least 20 years.” And in that time, thanks in part to this year's laureates, our understanding of the fundamental constituents of forces and particles has exploded.

15. NOBEL PRIZES

# Gold Medal From Cellular Trash

1. Gretchen Vogel

The cell's trash collectors, which control an internal system of protein disposal, are celebrated in this year's Nobel Prize in chemistry. The discoverers of this system, Aaron Ciechanover and Avram Hershko of the Rappaport Institute at the Technion-Israel Institute of Technology in Haifa and Irwin Rose of the University of California, Irvine, share the prize for work that established how a protein called ubiquitin, with several helpers, tags and delivers other proteins for recycling. The prizewinning experiments were “an extraordinary tour de force of classical biochemistry,” says Kim Nasmyth of the Research Institute of Molecular Pathology in Vienna, who helped clarify the role of ubiquitin in cell division.

While most biochemists in the 1970s were studying how cells make proteins, Hershko and Rose became interested in a less-studied puzzle: why a cell requires energy to break down proteins. In 1979, Hershko and Ciechanover, then a graduate student, pursued this topic with a series of experiments while on sabbatical at Rose's lab at the Fox Chase Cancer Center in Philadelphia. The result was a pair of papers published in 1980 in the Proceedings of the National Academy of Sciences revealing that proteins destined for destruction were covalently bonded—a process that requires energy—to a small protein the team called APF-1. That protein later turned out to be ubiquitin, which had been identified by other researchers a few years earlier, and which is found in eukaryotic organisms from yeast to mammals—hence its name.

The biochemists went on to show that three additional enzyme families, called E1, E2, and E3, work together to attach ubiquitin to proteins destined for disassembly. They and others subsequently showed that ubiquitin then delivers the doomed proteins to the proteasome, a large complex that breaks down the chemical bonds holding proteins together and releases their amino acid building blocks for reuse. Ciechanover says the discoveries honored by the Nobel committee helped explain how the protein-degrading proteasome can coexist with proteins in the cell's cytoplasm without breaking down the wrong ones. “The shark and the bait are living together peacefully, and they will interact only following the tag from ubiquitin,” he says.

A decade after the trio made their discoveries, researchers began to realize that ubiquitin's job was more than simple trash collecting. The protein and its enzyme helpers play a role in the cell's proofreading of newly minted proteins, targeting faulty ones for destruction. The ubiquitin system also helps regulate cell division, where it controls the swift buildup and breakdown of proteins that drive the cell cycle. It plays a crucial role in triggering DNA repair and apoptosis by influencing cellular levels of the tumor suppressor protein p53. And it helps regulate the signaling protein NF-κB, which triggers immune and inflammatory responses.

In recent years researchers have begun to piece together even more exotic roles for ubiquitin, including helping to transport proteins from the cell surface to the interior (Science, 13 September 2002, p. 1792). On the negative side, the protein is involved in enabling viruses such as HIV and Ebola to make their way to the cell surface after replicating inside the cell.

Drug companies also think they may find a way to exploit ubiquitin and its helpers. By blocking the system, researchers have been able to halt cell division in cancerous cells. One drug that blocks the action of the proteasome was recently approved for treating patients with multiple myeloma, a type of leukemia.

Nasmyth says the new Nobel laureates had no way of knowing how important their find would be. “This is a discovery that has impacted every single branch of biology and is a beautiful bit of chemistry,” he says.

16. NOBEL PRIZES

# Macroeconomists Showed Why Good Intentions Go Wrong

1. Charles Seife

It's no great insight to realize that governments behave in a less-than-optimal manner. Understanding why—that's another story. This year's Bank of Sweden Prize, otherwise known as the Economics Nobel, goes to Finn Kydland and Edward Prescott, two economists who figured out why good governments do bad things to good people. “I'm still high. It's a great event,” says Robert Lucas, an economist at the University of Chicago, who won the prize in 1995. “These are great economists.”

In the mid-1930s, economist John Maynard Keynes came up with a successful framework for analyzing broad trends in unemployment, consumption, production, and inflation. The Keynesian picture seemed to promise a utopia, a way to keep inflation and unemployment in check through an optimal strategy of setting taxes and interest rates and other tools of economic policy. But as with all utopias, an ideal economic policy turned out to be a pipe dream. Inflation and unemployment often fluctuated out of control, and occasionally a government's well-intentioned actions would make matters worse. Sometimes, the seemingly impossible would happen. For example, in the late 1970s, inflation and unemployment rose dramatically at the same time—something that the Keynesian picture forbids.

In the late 1970s and early 1980s, Prescott, of Arizona State University in Tempe, and Kydland, of Carnegie Mellon University in Pittsburgh, Pennsylvania, and the University of California, Santa Barbara, figured out why optimal-seeming fiscal strategies sometimes have suboptimal results. The two showed that governments have trouble committing to a policy; this lack of commitment leads to a credibility problem, which, in turn, can lead to an undesirable outcome. “The effect of a tax cut today depends on whether people think it is permanent or just temporary,” says Lucas. Inserting that insight into the mathematical models of macroeconomics changed the way economists think, he says: “It was a huge break from what all of us were doing at the time.”

“It just hit us in the nose,” says Prescott. The new approach also led to a better understanding of the causes of business cycles that rattle through an otherwise stable economy. As Prescott and Kydland discovered, it's in the equations: The best-laid schemes o' mice an' men gang aft agley.

17. # Behavioral Neuroscience Uncaged

1. Greg Miller
1. *8-13 August, Nyborg, Denmark.

Aided by dancing spiders, electric fish, and assorted frogs, neuroethologists continue a proud tradition of raiding nature's menagerie for insights into brain and behavior

NYBORG, DENMARK—All eyes were on the giant spider. Projected larger than life onto the screen at the front of the darkened auditorium, a sturdy specimen of the jumping spider Habronattus dossenus stared back with at least four of its eyes and fidgeted its hairy legs. Then it began to dance.

Male jumping spiders are renowned for their elaborate courtship displays, but Ronald Hoy, a neuroscientist at Cornell University in Ithaca, New York, was showing off a talent previously unknown for this arachnid. Aided by an amplified audio feed, Hoy demonstrated that the spider creates an auditory and seismic accompaniment to its visual display. As the spider strutted to and fro, scraping sounds and buzzes accentuated its movements, which were punctuated occasionally by a thump—the spider's abdomen slapping the ground—and a dramatic outward thrust of two front legs. Hoy told the audience that the spider's rhythm reminds him of flamenco, and a video comparison of the two dance forms brought murmurs of agreement.

Hoy's graduate student Damian Elias has found that the sound and seismic signals made by male jumping spiders are key to their mating success. Elias now plans to investigate vibration sensors in the spiders' legs that enable them to catch each other's vibes.

Hoy's presentation at the 7th International Congress of Neuro-ethology* illustrated a belief held passionately by the more than 500 researchers in attendance: Animal behavior—in particular, that of animals striving to survive and multiply in their natural environments—is fascinating to behold. Studying such behaviors and their neural underpinnings is the heart and soul of neuroethology.

At the congress, scientists from 19 countries presented work on a wide range of topics that have proven amenable to the neuroethological approach. The presentations included new findings on how the brain deciphers information gathered by the senses and also about the neural mechanisms of communication and movement control. Overall, the meeting's talks and posters, which featured a menagerie of creatures beyond the standard lab animals, illustrated how for more than half a century, neuroethologists have mined the tremendous diversity of behaviors and nervous systems in nature for general principles about how brains—including ours—direct actions.

## Neuro-what?

Neuroethology is generally not well known as a field, concedes Edward Kravitz, a researcher at Harvard University and incoming president of the International Society for Neuroethology, which organizes the triennial congress. The field traces its roots to pioneering studies in the first half of the last century by the likes of Karl von Frisch, Konrad Lorenz, and Nikolaas Tinbergen. Their work on the natural behavior of bees, birds, and fish highlighted the importance of interpreting animal behavior in the context of its survival value and showed that much could be gained by comparing behaviors across species. The fruits of this approach, which became known as ethology, earned the three a Nobel Prize in 1973.

Neuroethology goes one step beyond ethology to ask how the nervous system controls behavior. Its guiding principles include a bit of wisdom passed on by the Danish physiologist August Krogh, who suggested that for any question a biologist might care to pose, there is a species particularly well suited to provide an answer.

One way neuroethologists apply Krogh's principle is by studying species with exceptional talents. Owls are an oft-cited example. The birds hunt at night by localizing the rustles and squeaks made by their prey. By probing the auditory regions of owl brains, researchers have learned how the brain creates a spatial map from cues such as differences in the time it takes a sound to reach the two ears. This knowledge has clarified how less-expert species like ourselves localize sounds and, more generally, has illuminated how brains accomplish the seemingly impossible task of tracking events 1000 times more fleeting than a single nerve impulse.

“It's the specialist species more than the generalist species that tell us what biology can do,” says Hermann Wagner, who studies barn owls at the University of Aachen in Germany. When it comes to sound localization, he adds, “the barn owl is a Ferrari. … If you want to understand engines, wouldn't you rather study a Ferrari?”

Another application of Krogh's principle has led researchers to creatures with relatively simple nervous systems, which often happen to have exceptionally large—and therefore easily accessible—neurons. Studies of sea slugs, for example, have provided Nobel Prize-winning insights into how learning and memory modify the chemical communication pathways between and within neurons.

The second guiding principle of neuroethology is the comparative approach of the early ethologists. By comparing profiles of gene expression in the brains of songbirds with those of birds that don't sing, for example, researchers have begun to uncover clues about the evolution of vocal communication.

## A bird's-eye view

When Merlin the magician oversees the education of the young King Arthur in T. H. White's The Once and Future King, he turns Arthur into a fish, an ant, and various birds so that the young king will experience a variety of perspectives on the world. As that legend suggests, the sensory realm of animals is extraordinarily rich, and it has provided neuroethologists with fertile ground for study.

From a human point of view, one of the more foreign ways of taking the measure of the world is the sense of magnetoreception used by migratory birds to navigate between their winter homes and summer breeding grounds. The biological mechanism for the birds' magnetic compass has been a contentious topic for decades. In a presentation that generated animated coffee-break discussion at the congress, Henrik Mouritsen of the University of Oldenburg in Germany offered new experimental support for a rather startling hypothesis: that migrating birds literally see Earth's magnetic field.

A team from the University of Illinois, Urbana-Champaign, first raised that possibility in a theoretical paper published in the Biophysical Journal in 2000. It suggested that light-sensitive molecules called cryptochromes could form the basis of a biological compass.

The general idea is this: In response to light, crypto-chromes undergo a chemical reaction that creates so-called radical-pair intermediaries. A magnetic field, depending on its orientation, can alter the spin state of electrons in the radical pair and tweak the ratio of two final reaction products, which somehow turns the dial up or down on the chemical cascades that normally convert light to nerve impulses in the retina.

The implication, Mouritsen says, is that if cells in the bird retina contained cryptochromes, the planet's magnetic field would modulate the cells' sensitivity differently in different parts of the retina. “It might be that a bird perceives a ghost image of the magnetic field on top of whatever else it sees.”

Using fluorescently tagged antibodies, Mouritsen's team found that cryptochromes are expressed in certain cells in the retinas of migratory garden warblers but not in non- migratory zebra finches. The team also found that in the warblers, but not the finches, genes related to neural activity fire up in these cryptochrome-carrying neurons during magnetic orientation in the early evening—the time of day when wild birds take bearings from their magnetic compass (Science, 16 April, p. 405). Mouritsen recently published the cryptochrome findings in the Proceedings of the National Academy of Sciences.

The results look promising, says Wagner. “It's indirect evidence, and I think much more needs to be done, but it's the first solid clue that cryptochromes play a role in magnetic orientation,” he says.

And Mouritsen isn't the only one hunting cryptochromes. Andrea Möller, a Ph.D. student at Johann Wolfgang Goethe University in Frankfurt am Main, Germany, presented a poster at the congress that showed that crypto-chromes also exist in the retinas of migratory robins.

## Lines of communication

The way an animal perceives the world can determine the way it keeps in touch with its comrades. Consider electric fish, another favorite research subject of neuroethologists. The fish navigate through murky waters by creating an electric field and monitoring the field for distortions caused by obstacles. They generate the field by producing pulses with their electric organ, a modified muscle in the tail.

To navigate successfully, a fish has to keep track of its own electric-organ discharges and ignore those of its neighbors. Studying how they do this has revealed general principles about how animals sort through a barrage of conflicting sensory information and home in on cues that matter.

The fish also use their electric-organ discharges for communication. Harold Zakon of the University of Texas, Austin, has been investigating how and why these signals differ from fish to fish. His team has found that in some electric fish species, male sex hormones suppress the expression of genes for a particular component of the sodium channels essential for the electric organ's discharge. Channels built from these components turn on very quickly, enabling a rapid string of short pulses. Suppressing the genes for these subunits makes the males' pulses longer and reduces their frequency compared to those of females, an effect that's even more pronounced in the androgen-drunk males at the top of the social hierarchy. Variations in the males' signals may tell females who's the Big Kahuna, Zakon says.

Zakon has now extended this line of investigation to differences in communication signals among various species of electric fish. He and colleagues cloned sodium channels from 11 species of electric and nonelectric fish. All fish have two types of sodium channels, called Na1 and Na6, in their muscles. But in most electric fish, Zakon found, Na6 channels have been lost from muscle and are only expressed in the electric organ. And unlike the highly conserved sodium channels found in nerve and muscle, the Na6 channels in the electric organ appear to vary from one electric fish species to the next. The researchers identified several differences in the amino acid sequence of the Na6 channels that they suspect alter the way the channels open and close and account for the wide variation in communication signals seen in different species.

Keen perception and meaningful chatter are useless without the right moves to back them up. Understanding the neural control of movement is another major area of neuro-ethology research. Much of this work focuses on invertebrates. Crabs, slugs, leeches, and locusts have taught researchers a great deal about how circuits of neurons produce rhythmic behaviors such as walking, swimming, and flying. For many of these creatures, researchers have identified every neuron involved in producing a particular behavior, mapped out how one connects to another, and deciphered what chemicals they use to communicate—a level of understanding nearly impossible to achieve in more complicated nervous systems.

Such studies have clarified how sequences of electrical impulses from neurons generate the series of muscle contractions needed to bring about an intended motion. Two presentations at the congress, however, highlighted another aspect of controlling movement: the role of biomechanics.

Kiisa Nishikawa of Northern Arizona University in Flagstaff brought her colleagues up to date on her lab's work on how frogs zap tasty insects with their tongues. Over the last 20 years, she and her co-workers have studied nearly three dozen frog species and have identified three mechanisms of tongue projection. Their work has elucidated the evolutionary history of each mechanism as well as the interplay of nerve, muscle, and physics underlying all three.

In terms of brute force, the most impressive of the three mechanisms is called inertial elongation, which launches the tongue out of the mouth at 30 or more times the acceleration of gravity. The feat stretches the tongue to twice its resting length, substantially increasing the frog's range. This mechanism has evolved independently at least a half-dozen times, Nishikawa has found.

Coordinating this behavior would seem to be a complex task for the nervous system. It involves very fast, tightly coordinated movements of multiple muscles and joints. Yet Nishikawa's research has shown that much of the coordination that's required is built into the anatomy of the jaw and the physiology of its muscles.

Her modeling studies have revealed that more than 90% of the force for tongue projection comes from momentum transferred to the tongue from the lower jaw as it snaps open. The energy needed to launch the tongue is stored as elastic strain in a single pair of jaw muscles, and the anatomy of the jaw and tongue ensures that the tongue is flung forward on a remarkably straight trajectory every time.

The role of the brain in launching the tongue, therefore, is relatively simple. It's roughly analogous to the role of a medieval catapult operator: cranking the winch to increase the tension and sending the signal to let 'er fly. “Just because you have a complicated behavior doesn't necessarily mean you need a complicated control algorithm,” says Nishikawa.

This theme was echoed in a talk by Michael Dickinson, who described recent work in his lab at the California Institute of Technology in Pasadena on the flight of fruit flies. He and his colleagues have been investigating the quick turns that the flies make in midflight to avoid collisions. The team's studies indicate that all that's needed to accomplish these dramatic turns—90 degrees in less than 50 milli- seconds—is a modest tilt of the wing and a very slight change in the amplitude of each wing stroke. These adjustments are accomplished by steering muscles that alter the physical properties of the region of the thorax where the wing attaches (Science, 18 April 2003, p. 495). The brain can issue a few simple commands to the steering muscles, says Dickinson, “and what you get out is this beautiful maneuver.”

## Thinking outside the mouse

Asked to consider the future of their field, many neuroethologists see a mixed picture. On one hand, the field continues to attract young scientists and inspire new aproaches.

“We never have trouble getting students,” says Kravitz. Several postdocs have joined his lab, which studies the roots of aggression, after doctoral studies on fruit fly genetics. Kravitz says the researchers were elated to find that their knowledge of genetics could be applied to study behavior.

This sort of influx also brings fresh approaches to bear on traditional problems. For more than 20 years, Kravitz has studied how hormones and neurotransmitters mediate aggressive behavior in lobsters. Thanks in part to the expertise of the newcomers, the lab is now doing more refined experiments in fruit flies, using genetic tools to tinker with specific signaling molecules in specific neurons.

Other researchers are also realizing the power of genetics—using DNA micro- arrays, for example, to hunt for genes that rev up when a fish moves up or down the dominance ladder. Still others are drawing on computational advances. Speakers at one symposium at the congress discussed how robots can be used to test models of the neural control of movement—as well as how lessons from neuroethology might be applied to design more lifelike robots.

At the same time, many neuroethologists say that they've come under increasing pressure to justify their work, as governmental grant agencies have focused more on applied research and have sought a return on their investment in major genome projects. The situation has pushed neuroscientists toward using a few select animal models, says John Hildebrand, who studies moth olfaction at the University of Arizona, Tucson.

The trend is reflected in an “overwhelming emphasis on mammals” at large neuroscience meetings such as the behemoth annual gathering of the Society for Neuroscience, Hildebrand says. A search through the program of this year's meeting, for example, turns up 2593 abstracts containing the word “rat” and 3554 containing “mouse” or “mice,” but only 68 mentioning songbirds and 28 with electric fish.

Perhaps that's why many at the neuro- ethology congress say that the meeting has become a sort of refuge. “Every talk is like a celebration of a different model,” says Hildebrand. Like many attendees, Dickinson says the meeting has become one of his favorites: “At Neuroethology you don't have to hide the fact that you're really interested in behavior, and that's kind of liberating in a way.”