Introduction

Barely 15 years ago, only a handful of scientists in the military, medical, and veterinary fields had more than a passing interest in infections such as smallpox, anthrax, plague, and tularemia. Even fewer in the agricultural sphere worked on foreign pathogens that posed threats to U.S. livestock populations or our major crop commodities. Three federal government departments whose missions encompassed preparedness for uncommon infections—Agriculture, Defense, and Health and Human Services (HHS)—supported these efforts, and their combined budgets for this effort were small. But in 2005 talk of these infections is on many lips, the list of federal and state government agencies with active programs has grown significantly, and the funds available have mushroomed.

The purpose of this chapter is to explain how this came about and why the intelligence community and the Departments of Homeland Security, Justice, and State have now joined the traditional agencies—Agriculture, Defense, and HHS—as significant players in studies of infectious diseases of humans, animals, and plants. Subsequent chapters in the book explain in more detail the specific needs of these new entrants to the field.

This is not the place to recount the history of the development and use of biological weapons, which has been fully detailed elsewhere.1,2 The seminal events for our purposes occurred over the past 15 years; they are the realization that molecular techniques could transform the properties of microbial pathogens; discovery of the vast covert offensive biological weapons programs of the former Soviet Union; proliferation of biological weapons technologies to smaller countries that also harbored or supported organized international terrorist groups; attacks in Japan with biological and chemical weapons

Microbial Forensics

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mounted by Aum Shinrikyo, a religious cult with access to skilled scientists and abundant finance; attacks in the U.S. by domestic groups or individuals employing biological agents, such as the followers of Bagwhan Rajneesh in The Dalles, Oregon, and conventional explosives, as used by Timothy McVeigh in Oklahoma City, Oklahoma; and finally the al Qaeda attacks of September 11, 2001 and the anthrax letter campaign of October 2001 for which the purpose and perpetrator(s) remain unknown at this date. Today, we find ourselves locked in what appears to be a lengthy global struggle against a number of foreign terrorist groups at the same time that we confront individuals and groups domestically who are prepared to employ biological agents to advance their goals.

The common thread is that nation states, organized groups, and individuals can now see that for their own purposes it is possible to employ a group of readily available viral, bacterial, and fungal pathogens and their toxins— either in their native form or after genetic manipulation-to cause very serious consequences for humans, livestock, or plants. Questions of who would do this, why, and how have many answers—too many and too diverse to relate here.1,2 It is only important to realize that there are three concepts: biological warfare waged by nation states as an act of war; biological terrorism employed by terrorist groups that may or may not be supported by a nation state; and biological crimes (biocrimes) performed by individuals or small groups for nonwarfare or nonpolitical purposes.

Biological warfare—the use of disease-spreading microorganisms, toxins, and pests against enemy armed forces or civilians—as historically intended or conducted by nation states for military purposes, generally focused on infectious but noncontagious agents. These give rise to incapacitating or fatal illness in the human population exposed to the weapon, but pose no risk of rebounding on the attacker. Anthrax, tularemia, Brucella, and encephalitis viruses are the classic examples. The former Soviet Union's extensive development of smallpox biological weapons was an exception to this principle. Biological weapons targeting livestock or crops—such as the anthrax cattle feed cakes produced by Great Britain during World War II—were intended to deny the adversary's population the food supplies essential to continuing hostilities. We may still face the threat of biological weapons wielded by a nation state as an act of declared or undeclared war. But our military, political, and other means to counter such an event appear far greater than those we might employ against the much more dangerous yet amorphous threats of international terrorism.

The word terror, intense fear, is derived from the Latin verb terrere, meaning to frighten. There are many definitions of "bioterrorism." The one used here is: "the threat or use of biological agents by individuals or groups motivated by political, religious, ecological, or other ideological objectives."1 Aum

Shinrikyo provides an example; another may be the 2001 anthrax mail attacks. These events are different from the deliberate misuse of biological agents in biocrimes—as described in Chapters 4 and 18.

INFECTIOUS DISEASE: THE CLASSIC HEALTH VIEWPOINT

Biological agents developed as weapons pose different threats than the naturally occurring disease states traditionally associated with these infections in humans, animals, or plants. These differences become clear by a comparison of how public health or agricultural disease professionals and those engaged in creating offensive biological weapons programs see the infectious disease process.

Infectious agents, such as viruses, are composed of a delivery system—the outer coat of the virus—and a payload—the genetic material inside the virus. Their target is the host cell. This is a classic weapons scenario that those engaged in offensive biological weapons development know and exploit to the maximum. As Peter and Jane Medawar so aptly noted, "a virus is just a piece of bad news wrapped in protein."

The interaction between an infectious agent and a susceptible host is quite familiar, perhaps too familiar, to the medical professions and those charged with protecting animal and plant health. It is encapsulated in Figure 2.1: a microorganism is either virulent or not, and its contact with a host that is susceptible or not governs whether illness and death follow. The health professions in their entirety cannot help but look at this equation from one perspective—that the outcome be health and that the host regain full function, life, and happiness—because our education and ethical systems are built on these values.

Because of this perspective, health professions orient their research programs in infectious diseases along familiar lines: they dissect the infectious agent to understand those parts that might be used in a vaccine, and they examine the course of the infection in the host to find where the virus can be detected for diagnosis or where it persists in chronic infections. Their goals are simple: to identify the properties of the virus and of the host that can be of value for the purposes of diagnosis, treatment, and prevention.

Smallpox is by far the most important biological weapons threat because the disease is highly contagious, spreads easily by contact or aerosol from one person to another, and most probably could not today be limited to one country or continent after deliberate release. Variola virus, the cause of smallpox, can also be readily altered by genetic engineering techniques to enhance

Virus infection

Virus

•avirulent •virulent

Virus infection

Host Infected Host Outcome

• resistant •inapparent infection • recovery

• susceptible •clinical disease • death figure 2.1 Consequences of the encounter between an infectious agent and a host. If the infectious agent is not virulent or the host is not susceptible, there is no infection and no disease.

its virulence. The day is not far off when this pathogen can be synthesized de novo. An attack with the smallpox virus on the United States would threaten the entire world, could result in tens of millions of deaths, and could paralyze the economies of industrialized nations. Given these possibilities, health professionals have difficulty even comprehending that someone would deliberately release such a devastating disease. When forced to confront the threat of smallpox as a biological weapon, most health professionals would stress the need for adequate public health preparedness and see the most urgent questions to be answered as these:

• Do physicians have the skills and knowledge to recognize smallpox?

• Can the disease be diagnosed quickly and accurately?

• Do we have enough smallpox vaccine for the civilian population?

• Are first-responders vaccinated?

• Can we make a safer vaccine for immunocompromised individuals?

• Can we treat adverse effects of vaccination?

• Do we have policies and procedures in place for preventive and emergency vaccination?

Solutions to all these very important public health questions will certainly be critical if we should ever be attacked with smallpox, and the prudent first step as a nation must be to strengthen the public health system to answer each question positively. However, these are not the only important questions for a biological weapons defense program.

INFECTIOUS DISEASE: THE OFFENSIVE BIOLOGICAL WEAPONS VIEWPOINT

Scientists engaged in offensive biological weapons development see pathogens as weapons, and they look at the same equation for the interaction between a pathogen and a host shown in Figure 2.1. However, they do this from a very different perspective and with very different outcomes in mind from those involved in health care and delivery. For example, developers of offensive biological weapons want to understand the properties of the weapon's delivery system that allow it to spread by aerosol and survive in unfavorable environments, and those attributes of the payload that confer ability to evade host defenses and cause injury and death. They also seek to understand and undermine the properties of the target that confer resistance and susceptibility. Their goal is to enhance the weapon's capacity to cause injury in the host, and their desired outcome is death or disability. Understanding how a virus enters a susceptible target and how the virus outwits or evades the defenses of the host are critical factors in biological weapons design.

For those who want to make weapons, there are two desired outcomes: the native organism is honed to maximize death and injury, and advanced weapons based on the native pathogen are created by genetic engineering technologies. Advanced weapons have new properties, such as additional foreign genes that change the native biological properties to alter the species or organ system target, to overcome vaccination, to obscure diagnosis, to enhance transmission, or to add completely unexpected physiological effects. Advanced biological weapons pose new threats of technological surprise for the opponent. Wheelis3 outlines the issues in more depth.

The questions those engaged in offensive smallpox biological weapons development might pose include:

• Can smallpox virus be engineered to cause disease in people who have been vaccinated?

• Can another pox virus be engineered that has the same effects as smallpox and evades vaccination?

• Can this pox virus be made highly infectious by aerosol?

• Can the smallpox virus be altered to have additional unsuspected effects that would result in more disability and death?

• Can the recognition of smallpox by physicians or its detection and diagnosis be confused or delayed so infection could spread?

Were any of these questions to be answered affirmatively and such weapons released, our traditional public health defenses would prove insufficient. Given the past and present existence of covert foreign offensive biological weapons programs, the inexorable march of science and technology that could put these dangerous pathogens, knowledge, and skills into more and more hands, and the apparently growing ranks of non-state entities prepared to use such weapons, a comprehensive program to defend the U.S. must counter offensive biological weapons challenges and ensure public health preparedness. These are the factors that have brought new government players to the field.

INFECTIOUS DISEASE: NEW PLAYERS AND NEW THREATS

Department of Defense and the Intelligence Community

These agencies have always had the prime roles in defense against traditional biological warfare and have vigorously responded to changing threats over the past 15 years.

Defense focused first on real-time detection of biological weapons agents in air, water, and soil on the battlefield and translating detection into immediate effective protection of the Warfighter. Those biological agents long considered potential weapons were the first priorities. As the nature of the threat to our armed forces has become more diverse—all those persons on military bases at home and abroad, critical ports through which troops and material must load and unload—so Defense programs have expanded to provide a broader range of detection and countermeasures against a broader array of pathogens that might be encountered on and off the traditional battlefield.

The roles of intelligence community members will not be discussed in detail here. Suffice it to say that entities such as the Central Intelligence Agency are charged with using the equation shown in Figure 2.1 to discover those posing threats to us abroad, the nature of these threats, and the potential targets and means of delivery. Biology is just one of the technologies deployed for these purposes. The reader will appreciate the singular challenge posed in trying to identify a few people engaged in threatening activities among a global ocean of legal activities employing almost exactly the same types of persons, equipment, and facilities in academia, industry, and government. And even when suspects are identified it may not be possible to gain entry to the facilities they use, and thus remote means must be employed to discover what is going on inside. The kinds of biological questions the Agency might face could include:

• What pathogen is in the facility?

• Can that pathogen be reliably recovered from and identified in nonclinical materials from the suspect facility?

• Is reliable recovery possible after materials have been treated in ways adverse to pathogen survival?

• How few copies of the pathogen can be there for detection and how much of the whole organism is essential?

• Where did the pathogen come from?

• Has the organism been genetically altered and, if so, what unexpected properties have been added?

• Is there a foreign gene(s) present in the pathogen?

• Is there any other evidence from the pathogen, the genome, or the delivery vehicle that would indicate laboratory origin or deliberate manipulation?

Department of Justice and the Federal

Bureau of Investigation

Deliberate attacks with biological weapons on the U.S. or U.S. interests at home and abroad would be either an act of war, if conducted by a nation state, or a crime. In either case, the Federal Bureau of Investigation (FBI) would be called upon to investigate. The FBI must thus look at Figure 2.1 with an eye to preparing for a wide variety of investigations: to determine what can be gleaned from studies of the biological agent, the victim and, perhaps, the perpetrator, to clear the innocent and to identify those responsible so that they may be successfully prosecuted.

This volume is largely devoted to the FBI's scientific needs for microbial forensics—the tools and technologies that will equip the Bureau to investigate biological crimes successfully. That such specialized and unmet needs exist at all will come as a surprise to the vast majority of readers, and the first purpose of this volume is to alert the wider scientific community to this deficiency. The chapters by Budowle and colleagues and by Harmon explain what the Bureau needs, how biological information will be used with other evidence to investigate and correctly attribute and prosecute criminal activity, and the standards that courts will demand for the scientific evidence. The rest of the volume starts to outline the field of microbial forensics through example and application of specific technologies. We intend that the volume as a whole should fulfill a second and greater purpose: to attract other scientists with novel ideas and skills to the field.

Department of Homeland Security

The FBI is charged with investigation of potentially criminal activity involving biological agents, during which the Bureau will use tools mostly developed by other agencies, of which the Department of Homeland Security (DHS) is the principal. The chapter by Budowle and colleagues explains the role of the DHS's Bioforensic Analysis Center as the core of a coordinated national activity.

Department of State

The Department of State would take the lead in responding to biological attacks on U.S. citizens and interests overseas—other than those occurring as an act of war, which would fall under the aegis of the Defense Department. In addition, State is responsible for monitoring compliance with the Biological and Toxin Weapons Convention. To meet this mission, State must be capable of identifying suspicious disease outbreaks in humans, animals, and plants overseas that might be indicators of covert or illegal biological weapons activities undertaken by a nation state, organized group, or individuals. This is not a simple or rapid task, as illustrated by the Sverdlovsk anthrax outbreak described by Budowle and colleagues elsewhere in this volume. At a minimum, State must look at Figure 2.1 and be able to:

• Define how suspicious human, agricultural, and wildlife disease outbreaks differ from natural disease outbreaks in terms of surveillance, diagnostics, characterization, and attribution.

• Identify any special measures, qualifications, procedures, safeguards, technologies, or scientific approaches that must be used in investigating such outbreaks given the limited capabilities of established international human and agricultural health agencies.

• Field an effective investigative team that is backed by proven microbial and biological forensic techniques that can meet the scientific evidence requirements of international courts.

Currently the U.S. cannot provide these capabilities to the extent one would desire: the DHS's Bioforensic Analysis Center and the FBI will be leading U.S. capability- and capacity-building in the necessary areas. Nor are there international agencies that might fulfill these needs. International agencies monitoring livestock and plant health matters have very limited capabilities, and those for wildlife diseases are virtually nonexistent. Even the World Health Organization (WHO), probably the best resourced of any, maintains a healthy distance. A recent report4 outlined that agency's anticipated role in any inci dent of intentional, malevolent use of biological or chemical agents. Key points included:

• WHO will focus on the possible public health consequences of such an incident, regardless of whether it is characterized as a deliberate act or a naturally occurring event.

• WHO advises strengthening public health and response activities with an emphasis on:

0 More effective national surveillance of outbreaks of illness 0 Better communications between responsible agencies and better coordination of their responses 0 Improved assessments of vulnerability and effective communication about risks to both professionals and the public 0 Preparation for handling the psychosocial consequences of the deliberate use of pathogens and chemicals to cause harm, and 0 Contingency plans for an enhanced response capability

WHO also points out that: "Should the United Nations be called on to respond to a request to investigate, WHO could be asked to provide technical expertise or to make available its existing resources and mechanisms. Non-public health issues related to investigations of reports on possible use of chemical and bacteriological (biological) or toxin weapons, however, remain the responsibility of the United Nations."

Clearly, the Bioforensic Analysis Center and FBI will be meeting an important need for the U.S. and setting a key precedent for the international community by fulfilling the research and development program outlined elsewhere by Budowle and his colleagues. This volume will be critical in informing the broader U.S. and international scientific communities of the opportunities for contributions in this important area.

Specifically, in terms of future research investment, attention should be drawn to a significant gap in our knowledge of disease agents in wildlife species, in which category I include biting insects and ticks. This is an area in which suspicious disease outbreaks should be expected, especially at habitation borders where humans are now encountering wildlife species—and pathogens—from which they were previously separated by geography.

In terms of known pathogens, this would include most of the top human threats (except smallpox), such as plague, tularemia, anthrax, botulism, hem-orrhagic fever viruses, encephalitis viruses, Nipah and Hendra viruses, and other zoonotic pathogens, several of which are transmitted by mosquitoes, ticks, and other biting insects.

Wildlife species, including biting insects and ticks, are also the reservoir of many unknown infectious agents that may cause little or no illness in the wild species which have adapted to them by evolution. However, when these infectious agents enter a novel host, such as humans, they can provoke very serious and fatal disease. Examples include: Ebola virus (possibly in a chimpanzee reservoir), Hendra and Nipah viruses (maintained as inapparent infections in fruit-eating bats), and Hanta virus (maintained in mice). The interface between wildlife and humans is an area of great risk to human health normally, and is one of the most likely sites for future suspicious disease outbreak investigations. Incidents of fatal human diseases caused by presently unknown pathogens can be predicted in Africa and South America as people begin to live permanently on newly cleared land at the margins of virgin jungle where they are exposed to biting insects and other wild reservoirs of these infections. Paradoxically, however, in the past ten years, Hanta virus occurred in the American Southwest and Hendra and Nipah viruses appeared in Australia and Malaysia, respectively. The latter two viruses, representative of a new family of viruses whose other members we do not yet know, were carried by fruit-eating bats that had no direct contact with mammals: the infection was transmitted by feces from bats roosting over places where horses or pigs were housed.

It is worth noting that the unknown pathogens that cause serious diseases and death in wildlife species will likely not be detected or raise alarm by themselves. The trigger for attention will be when these pathogens spill over into humans (Hanta virus), or horses and then humans (Hendra virus), or domestic swine and then humans (Nipah virus). The tools and technologies to investigate disease in wildlife species will thus be developed primarily to investigate disease in humans or livestock caused by the same pathogens.

INFECTIOUS DISEASE: WHAT ARE THE THREATS?

There are many dangerous pathogens that could be used in deliberate attacks on humans, livestock, and plants. A question that is often asked is: "Which is the most important?" The answer is: "The next one to be employed against the U.S." This is not a trite comment. Because several federal agencies are struggling to balance a finite amount of resources against a growing list of potential threats, we cannot approach this problem linearly.

There are various "lists" of priority pathogens, and these vary by the originating agency. Defense has a fairly short list of pathogens that can mostly be traced back to a historical state-supported offensive weapons program. HHS has a somewhat longer list that adds domestic terrorism precedents, such as ricin and Salmonella, and foreign viral diseases such as Rift Valley fever and

Nipah virus. Agriculture maintains a long list of animal and plant pathogens, but how much of a threat most of these pose for the U.S. awaits more critical analysis. For example, it is difficult to envisage how the U.S. could be terrorized by camel pox, a virus that infects only that species. In any event, in the case of foreign animal and plant diseases, it is only the ways we have chosen to respond to inadvertent disease introductions in the past—by mass slaughter of infected hosts and sweeping trade penalties—that enable terrorists to threaten us with them in the future.5

But however the lists are chopped and changed, it is clear that agencies such as the FBI and State are always likely to be faced with suspicious incidents that involve either unknown pathogens or pathogens for which the specific science base is not available. As a result, analytical techniques that can be developed and used "on the fly" will always be necessary.

INFECTIOUS DISEASE: BIOSAFETY AND BIOSECURITY

Those who work with infectious agents are familiar with biological safety standards developed by the Centers for Disease Control and Prevention to protect the health of laboratory staff by reducing or eliminating accidental exposure to human pathogens and to prevent the release of microorganisms from the laboratory into the environment.6 These standards are based on facilities constructed and operated so as to contain the pathogens and on working practices in the laboratory that safely manage infectious agents. Many scientists thought these guidelines were stringent enough to prevent release of dangerous pathogens from the laboratory, but they did not consider the possibility of deliberate theft. During 1999 and 2000, informal attempts by government scientists to develop specific security standards could not achieve interagency consensus. The absence of precedents of theft and malicious use undermined attempts to make security a priority, even though in this regard many laboratories handling dangerous pathogens stacked up poorly against supermarkets and shopping malls.

The anthrax mail attacks in fall 2001 ushered in extensive security restrictions in all laboratories handling "select agents," a specific list of high-consequence pathogens defined by the Departments of HHS and Agriculture. These restrictions involved physical security standards, personnel surety programs, and tighter controls on inventory, shipment, and transport of pathogens. To the traditional base of "biological safety (Biosafety)," these new laws added another layer of "biological security (Biosecurity)," designed to protect biological agents against theft or sabotage. As a result, the biological research community has chafed under the unfamiliar burdens of operating in a security-conscious environment. These burdens are unlikely to lift soon given increased public awareness of biological weapons and the threat of terrorism, and a belated realization that diagnostic and research laboratories are potential sources of viable, virulent pathogens and their toxins. Nevertheless, there is no doubt that in the rush to do something about security, most relied far too heavily on guns, guards, and gates.

The biological community needs specific new tools to achieve a balance between adequately monitoring certain biological agents and toxins (far fewer than are on the lists today) and not obstructing legitimate research and development involving those agents and toxins. A good place to start would be a reappraisal of the biological agents subject to restriction: not all agents need the same level of scrutiny, and the ones requiring the tightest safeguards should be those that are most attractive to terrorists because they are the most devastating when used as weapons. The next step should be a proper Biosecurity Risk Assessment that starts with a definition of the facility's biological inventory and evaluates the consequences of its loss, a step that enables the pathogens to be prioritized based on those consequences. The identities of those who might seek to steal the assets, along with their motives and methods can then be assessed. Then the overall risk—probability and consequences— of these undesirable events can be evaluated.

Microbial forensics in support of law enforcement must become a critical new tool. Specifically, if we can move beyond simple statements about lists of microorganisms possessed by a particular facility to the stage where each isolate in the inventory is characterized and "fingerprinted" (bearing in mind the limitations of this analogy described elsewhere in this volume), we will be able to move quickly in an emergency to differentiate those who are definitely not involved from those who could be. For example, the DHS's Plum Island Animal Disease Center is the only facility in the U.S. legally allowed to possess certain high-consequence livestock pathogens, most of which are viruses. In the event of an outbreak of one of these diseases in the U.S., the first suspicion today would not be an inadvertent introduction but a terrorist act. The second thought would be that someone at Plum Island might be involved. At the very same time that the U.S. would need the services of its handful of experts, they could be under suspicion. The ability to "fingerprint" immediately the outbreak virus and compare this with the fingerprints of those viruses stored at Plum Island would resolve this conundrum. Clearly, for the vast majority of biological laboratories and pathogens, the issue is not so simple. However, we can apply the same principles to a whittled down list of high-consequence pathogens, given application of the technologies described in this volume. There is a long way to go, but the road ahead is clearer than ever before.

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