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Originally published in the Summer 2002 Virginia Tech Research Magazine.

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Ann La Berge was reading 19th century French newspapers at the time of the anthrax outbreak in the United States. When she received a package that had been shipped through the Brentwood post office, she threw it out and washed her hands.

“I was reading about the cholera epidemic,” says La Berge, a Virginia Tech historian of medicine and faculty member in the science and technology studies program. “Government officials and physicians were being quoted as saying, ‘There is nothing to worry about. We are the most civilized nation in the world. What is happening in other places will not happen here.’ Then, within a few months, 18,000 Parisians out of a population of 750,000 died of cholera.”

In 1832 Paris, people did not have radio, TV, and the Internet to keep them informed, and literacy was low so the newspapers were not widely read. But they had startling personal experiences. “They would see people collapse in the street. They would go out to dinner and three days later learn that a dinner companion had died. There were bulletins and posters with updates on who had died and how many had died. And there was rumor — which was much scarier than today because it was more difficult to verify,” says La Berge. “Among the poor, the rumor developed that the disease was government sponsored to eliminate the poor, who were becoming a burden. Conspiracy theories were always around: ‘The government knows what is happening and they won’t tell us.’”

In 21st century America, government officials want to preserve the national order and want the public to think the appropriate agencies are on top of the situation, La Berge says. “But when someone says, ‘There’s a slim chance,’ I take it seriously when there is something I can do.”

Thomas Toth continued to open his mail. A virologist in the Virginia-Maryland Regional College of Veterinary Medicine, he teaches a course on emerging diseases, including ones that are chilling indeed, such as Ebola.

“When you have anthrax, you have signs and symptoms you can recognize and respond to,” he says. “Anthrax is not easy to use as a bioterrorism agent. It needs sophisticated production facilities. It’s not contagious from person to person.”

Thomas Inzana, a bacteriologist and director of clinical microbiology and coordinator of the Center for Molecular Medicine and Infectious Disease (CMMID) at Virginia Tech, agrees with Toth. “Diseases due to Neisseria gonorrheae and bacterial meningitis are much more common than infections
due to all the bioterrorist agents taken together,” says Inzana. “A college student has a better chance of getting meningitis and an elderly person has a greater chance of developing pneumonia.”

La Berge, who has studied 19th century public health for 30 years, actually agrees with Toth and Inzana. “Anthrax and smallpox are problems we thought we had solved. Today, there are other concerns — obesity, AIDS ... But epidemiology is not only the science of microorganisms.”

History of disease

The impact of epidemic disease on history has been significant, she says. “A small number of Spaniards were able to conquer the Inca and Aztec nations because of the unwitting introduction of microorganisms against which
the Indians had no defense.”

In the middle ages, the Black Death (bubonic and pneumonic plague) killed 33 to 40 percent of the population in some places in Europe. Attempts to track the source created lively folklore, with everything from planet alignment to religious affiliation being blamed.

During the Civil War, most deaths were not battle deaths, but were caused by disease. “Microorganisms have been major historical actors,” La Berge says.

Modern epidemics include influenza in 1918, which killed more than 20 million people worldwide. “In the 1918 flu epidemic, almost no one was untouched,” says La Berge. “My uncle lost both his parents and he and his siblings had
to live with other relatives. It was easily spread. It was the sort of disease that could wreak havoc.

“During the polio outbreaks of the 1950s, people who could stayed away from public places, such as theaters or swimming pools — anywhere there were crowds. Some young men came home from a world war only to be struck down by polio. Although the numbers of people infected were not great, the threat was widely publicized. So a vaccine was developed.”

There are parallels with the anthrax outbreak, she says — low numbers, publicity, work on a better vaccine.

The government response appears largely unchanged in 50 or 100 years. “The aim is to keep people calm, maintain public order, and maintain the infrastructure. Otherwise, the public response could be as bad as the disease, with widespread job loss and dislocations,” says La Berge.

Focus on preparation

However, there is a difference in response. The focus is now on disaster preparedness.

“The government response now has more to do with reality than wishful thinking,” says Toth. “It is why we have the Centers for Disease Control and Prevention (CDC).”

“Disasters have phases,” says Taranjit Kaur, the university veterinarian at Virginia Tech, whose research includes preparing for and responding to biological disasters. In the case of a natural disaster, there is a non-disaster phase, a pre-disaster phase (“when you are apprised that a cyclone is coming or the river is rising”), an impact phase, response phase, and recovery phase.

“With a biological disaster, you are not warned and the impact is insidious,” she says. “You may not know of the impact until people are in the hospitals, exhausting and perhaps contaminating the medical infrastructure, reducing our ability to respond. The community is impacted before it knows there has been a disaster.”

The longest phase in the disaster cycle is the non-disaster phase, which is when disaster preparedness must occur, she says. “You need the fire drills so the mind will kick in based on pre-conditioning.” Simple public health protections that do not rely on bigger systems are important, Kaur says. A number of agencies, including the Humane Society of the United States and a number of universities, already have disaster plans that others could adopt with some modifications, or use for training purposes.

And, public information is important, Kaur says. “Aerosolization to spread chemical or biological agents is a possibility, yet some officials would hesitate to advise people to stay indoors or cover their mouth and nose, believing it will only cause panic. But people need to know how to respond to a biological threat. The goal is to prepare the general population. It is important to take whatever precautions we can take as individuals.”

Vigilance and identification critical

A new level of vigilance is required, she says. “The challenge is recognition. In the case of disease agents that medical staff are not trained to recognize, there is a lag time in recognition. What appears to be an unusually high number of cases of flu in the elderly community may be something else. But medical staff may have to become ill themselves before there is an attempt to identify a microorganism. All medical personnel should have a heightened awareness to the possibility of exotic causes of disease.”

Animals can be sentinels for infectious disease, she says. “When the birds in the Bronx Zoo were showing evidence of disease, it was an early sign of West Nile Virus. But public health officers were not sensitive to this as an early indicator of infectious, potentially zoonotic, disease.”

Medical professionals also need to be more routinely cautious. “The fact that more members of the medical community are wearing gloves because of HIV helps, but face masks are still not standard practice, even for emergency workers.”

Given biological agents’ capacity for mass distribution, it is crucial that there be rapid identification of the attack agent, identification of the individuals exposed, and implementation of treatment. “The response and recovery will, to a large extent, be contingent on the rapidity and accuracy with which the biological agent is identified,” says Kaur.

“Now we have technology that allows us to see genetic information within a day that it took months to find just 10 years ago,” says Toth. “Our tools are more efficient, sensitive, and rapid. Look at the highly lethal infectious disease that occurred in 1993 in the Four Corners area of New Mexico. The fact that it was caused by a new variant of a long known Hantavirus was determined within three weeks. Just a few years earlier, it might have taken months.”

At Virginia Tech, researchers in biochemistry, chemical engineering, and physics are working on developing supersensitive, multi-agent biosensors that can withstand the rigors of the field. Meanwhile, many scientists are developing diagnostic tests for specific pathogens and working on vaccines. Among the many diseases being targeted at Virginia Tech are terrorist agents, such as anthrax and brucellosis, and several common killers and emerging diseases.

Lethal weapon

Inzana is studying tularemia, a particularly virulent disease that has already been developed for warfare. “The clinical manifestations are similar to many other infections, including plague, and it only requires 10-50 organisms to make you sick,” he explains. “By comparison, anthrax requires at least 5,000.”

Tularemia can be worse than plague because in addition to being transmitted by parasite vectors, it is also transmitted by water, tissue of infected animals, food, the environment where infected animals have been, and various flies. “The organism gets inside the white blood cells — the ones that are suppose to kill it,” says Inzana. “The immune response to the infection can cause lesions in the host’s organs.”

The Virginia Tech research is focused on the polysaccharide capsule, the cell component formed of sugar molecules that surround many bacteria. “The objectives are to purify the capsular polysaccharide (CP), raise immune serum to the CP, and to develop rapid field diagnostic tests to detect the CP, and hence the organism,” says Inzana. “The diagnostic test will be sent to colleagues at the CDC to determine if all strains of F. tularensis contain an antigenically identical CP. If so, work to develop a subunit vaccine will also be undertaken.

“We will create a vaccine with the capsule conjugated to a protein from the cells’ surface to initiate antibodies and a cellular response within the potential host,” he says. “The strategy is to kill the organism before it gets into the white blood cells, and to avoid using whole cells and live vaccines.”

Common killers

CMMID researchers are also developing a pathogenhost model for human diseases for which the only natural host, humans, cannot be used. Such diseases include N. gonorrheae and Haemophilus influenzae.

H. influenzae can be spread by contact with infected respiratory secretions and is one of the causes of bacterial meningitis, pneumonia, and middle ear infections. Gonorrhea is a sexually transmitted bacterium that causes disease in hundreds of thousands of people in the United States each year. It may cause systemic disease as well. Both of these organisms can change the make-up of carbohydrate antigens on their surface with a molecular switch that turns the gene on and off, a process called phase variation.

H. influenzae and gonorrhea are genetically similar to Haemophilus somnus, which can cause meningitis, respiratory, and urogenital diseases in cattle. H. somnus uses the same type of genetic switch to phase vary its components as H. influenzae and N. gonorrheae. Understanding how an animal pathogen uses phase variation to evade host defenses in its natural host should also apply to a human-specific pathogen.

The CMMID researchers are working with scientists at the University of Oklahoma Health Sciences Center and the Virginia Bioinformatics Institute at Virginia Tech to sequence the H. somnus genome. They expect to annotate or understand the function of the proteins that result from specific sequences by early 2004. “H. influenzae has already been sequenced and annotated, so we will be able to determine how many of the genes in the two organisms are the same. We will then be able to determine what proteins are expressed under different conditions and sites in the natural host,” Inzana says.

Focusing on the gene sequences shared by the human and animal agents, “We will see what effect modifying specific H. somnus genes has on the ability of the bacteria to infect its natural host. Strategies that control H. somnus should have relevance to the bacteria that infect humans,” Inzana says.

It wouldn’t be the first time that investigation of a cow disease helped to prevent a human disease. More than 200 years ago, people took advantage of the fact that exposure to cowpox resulted in immunity to smallpox. The term vaccination comes from the Latin vacca or cow.

Inzana, who is a clinical microbiologist in human microbiology, explains that there are many causes of bacterial meningitis and pneumonia, and for many agents vaccines are still not available. “There is a vaccine available to prevent meningitis due to one encapsulated type of H. influenzae, but not against pneumonia and ear infections due to non-encapsulated strains.”

Bacteria that cause pneumonia often live in the human upper respiratory tract, such as the nasopharynx. “They are not a problem until they get into the lower respiratory tract or if the immune system is compromised, which is why influenza in the elderly can result in pneumonia,” Inzana says. “This scenario also occurs in animals, which is why a lot of our focus is on animal models.”

Meeting mutations

What about staying ahead of mutations? “The flu is a good example. It is a global infection. Birds are the common host, but virulent strains have gone through pigs, and may later infect humans,” says Inzana. “In the United States, we can see what strains are occurring in other parts of the world and determine how a new strain differs from a previous strain.”

Antigenic drift is a slight change in the arrangement of the genetic material or composition of the surface antigens, whereas antigenic shift is a major change and enables the bug to elude detection by the host’s immune system. “The new bug is compared with the old bug to make a new vaccine.”

The 1918 flu epidemic was a result of a major antigenic shift. No one had immunity, Inzana says.

The work done to stay ahead of mutations so we can have a flu vaccine is similar to the work CMMID researchers are doing to use animal models for human disease. “In bacteria, we look at antigenic phase variation between the disease organisms that infect animals and those that infect humans,” says Inzana.

It came from the barnyard

Some of the most serious diseases are the ones that come from animals. “We are all hosts,” says Toth. “Some diseases are specific to animals and some to humans. Many are zoonotic — they can travel between the two.”

Among the zoonotic diseases that Virginia Tech is working on, one is ancient and one is a newly discovered threat to humans.

Brucellosis probably already infected cattle when they were first domesticated. Now, thanks in part to the vaccine, RB51, developed in 1996 for Brucella abortus by Gerhardt Schurig and colleagues in the College of Veterinary Medicine, brucellosis has been essentially eradicated from U.S. cattle herds. The vaccine is now being adopted for use in livestock worldwide.

In 2000, Stephen Boyle, professor of microbiology; Schurig, professor of immunology; Nammalwar Sriranganathan, associate professor of microbiology; and their pre and post-doctoral trainees developed an attenuated vaccine that is a modification of RB51. It lacks some of the sugars on the brucellosis lipopolysaccharide envelope so it is not virulent. The newer Brucella livestock vaccine includes antigens for tuberculosis, paratuberculosis, neosporosis, Rift Valley fever, and other livestock diseases.It is the first time an attenuated multivalent vaccine for these diseases is being tested in animals.

With funding from the Department of Defense, Sriranganathan, Boyle, and Schurig are also pursuing research to use the Brucella RB51 platform for a human vaccine against anthrax and other human diseases, including TB. In the case of anthrax, the researchers have synthesized portions of anthrax DNA and inserted it into strain RB51. The aim is to create a vaccine that confers greater immunity with fewer side effects than the present vaccine. The DNA sequence codes for the “protective antigen,” which is not the cause of the disease but is essential for the functioning of the toxic factors. Several vaccines are presently being tested in laboratory animal models.

One new zoonotic threat, only recognized in 1988, is the hepatitis E virus (HEV), which causes human hepatitis E. A virus carried by swine, and less commonly by rats, dogs, chickens, and possibly other animal species, HEV can be passed to people through fecal contamination of water and water supplies or direct contact with infected animals. HEV causes acute illness and is fatal to 15 percent or more of infected pregnant women.

It is not related to other (A B, C, or D) hepatitis viruses. The term, “hepatitis” simply means the virus targets or damages the liver. The initial site of HEV replication in pigs or humans is unknown. The mechanism by which the virus reaches the liver is not understood either. A vaccine against HEV is not yet available.

HEV was not thought to occur in humans in the United States. But, in 1997, Xiang-Jin Meng of Virginia Tech and colleagues at the National Institute of Allergy and Infectious Diseases discovered a strain of HEV in U.S. pig populations and detected HEV antibodies in the blood of about 20 percent of blood donors. More recently, Meng and postdoctoral associate Reza Haqshenas have discovered yet another new hepatitis virus in chickens, designated avian HEV. Like swine HEV, avian HEV is also genetically related to human HEV.

'The most effective strategy when planning disaster preparedness programs is developing an infrastructure to manage the worst case scenario - and biological disasters are viewed as the worst case. ... A critical need is an assessment of diagnostic resources.'

Meng, a molecular virologist in the College of Veterinary Medicine and a trained physician, has demonstrated that human HEV infects pigs and that swine HEV infects such nonhuman primates as chimpanzees and rhesus monkeys. His group also showed that pig handlers are at higher risk of HEV infection. Funded by the National Institutes of Health, he is developing better assays to detect HEV infections, looking at the risk of cross-species infection and the threat to xenotransplantation (use of organs from genetically engineered pigs in humans), determining whether swine can be a useful model for the human virus, and is working toward the development of a vaccine against HEV infection. In collaboration with his CMMID colleagues F. William Pierson and Toth, Meng’s group is also investigating whether the newly discovered avian HEV from chickens can also infect humans.

HEV is one of scores of pathogenic microbes that have only been recognized in the last 30 years — some because incidents of illnesses are increasing and the technology now allows us to identify the microorganisms, and some, such as Ebola and HIV, because they have only recently become zoonotic.

On high alert and committed to battle

Advancements in public health, diagnosis, and treatment have been logarithmic, says Inzana. “But, when you conquer one disease, another fills the niche.”

“Forget about beating disease. It won’t happen,” says Toth. “But we can achieve a balance. Some diseases are in decline and some are up and coming.”

“But now we have to calculate the potential of bioterrorism,” says La Berge.

“With the advent of serious bioterrorist threats, prediction/prevention is critical,” Bruno Sobral, director of the Virginia Bioinformatics Institute at Virginia Tech, told the U.S. Senate Subcommittee on Science, Technology, and Space in February. “For many years, defense specialists have used a technique called scenario-building to anticipate and plan for even the most unlikely circumstances. The most successful results are achieved by bringing together thinkers and doers from diverse perspectives.”

Kaur has also been recommending that such a committee be formed to define the worst case scenario, develop a strategy to network resources, define operating procedures, and implement training and fire drills. “The most effective strategy when planning disaster preparedness programs is developing an infrastructure to manage the worst case scenario — and biological
disasters are viewed as the worst case at this point in time,” she says.

A critical need is an assessment of diagnostic resources, she says. “There are only two known laboratories in the United States capable of rapidly and accurately diagnosing agents of biological weaponry — the CDC and the U.S. Army Medical Research Institute of Infectious Diseases. But other laboratories also have developed some capacity. An assessment of the existing resources is needed, specifically, state and local public health labs and diagnostic labs at all hospitals in major cities.”

The Virginia Bioinformatics Institute (VBI) is an example of a new kind of resource. The institute provides genetic sequencing of pathogens as needed, “but our primary mission is to create a single bioinformatics interface to access the already available information required for a comprehensive surveillance program,” Sobral told the Senate subcommittee. “We integrate (data) and provide molecular information regarding pathogens, their hosts, and their interactions within the environment.

“The completion of the information pipeline — from basic research, to data interpretation, to useable information, to knowledge, to applications and technologies — requires a strengthened partnership between government, academe, and industry,” he said.

In addition to Virginia Tech, VBI has a partnership with the Johns Hopkins Bloomberg School of Public Health, which expands the study of major infectious diseases and provides field data to integrate in the pathogen database. Partnerships with industry have provided the necessary technology for integrating information. “Infectious disease is global, as HIV, malaria, and foot-and-mouth disease prove. Early identification and intervention are pivotal,” Sobral said. “We will create a common source of fundamental scientific information that has been fragmented to date. Integration on this new level will promote proaction rather than reaction.”

“For each of the biological weapons considered by experts to be the most serious threats to America — anthrax, botulism, plague, smallpox, and tularemia — modern medicine has some effective means of responding, whether by vaccination, antibiotic, or antitoxin. To inhibit the spread of a biological attack or a ‘normal’ disease outbreak in humans, livestock, or crops, we must have rapid diagnostic tools, a public health system to track disease as it evolves, and epidemiological data to determine the origin,” Sobral told the Senate subcommittee. “Fundamental research and expertise provided by universities will be essential to complete these tasks. It will provide the foundation to deliver the tools with which we will prevent, detect, protect, and treat victims of biological terrorist attacks.”