Biological warfare has existed for centuries, with one of the earliest known examples occurring in 1155 when Emperor Frederick Barbarossa poisoned water wells with human bodies in the siege of Tortona, Italy. In 1972, the Convention on the Prohibition of the Development, Production, and Stockpiling of Bacteriological (Biological) and Toxin Weapons and their Destruction was signed and adopted by the United Nations Office for Disarmament Affairs for enforcement. The treaty aims to prevent the development of offensive biological weapon (BW) agents and eliminate existing stockpiles; however, it only applies to the 170 nation-states that signed the convention and does not affect the actions of the 23 non-signatory states, such as Chad, Israel and Kazakhstan, or independent groups and individuals that seek to employ such weapons.
The 2001 anthrax letters in the United States demonstrated that the 1972 BW convention limits only one aspect of the problem. Weapons of mass destruction (WMD), once previously under the sole control of nation-states, now could be maintained and deployed by an individual. In 2010, it was concluded that these letters, which were mailed to political leaders and media outlets across the United States, constituted a terrorist attack and were sent by Dr. Bruce Ivins, a trained microbiologist employed by the U.S. Department of Defense.
Another set of biological attacks occurred in April and May 2013. Two separate ricin letter attacks were allegedly carried out by individuals who, with little to no scientific experience and support, were able to create a biological agent, albeit one that may not have had the potency of an effective weapon. Compared to the 2001 anthrax letters, the 2013 ricin letters illustrated a transition in BW production from the trained individual to the layman, as it has been alleged that the first set of letters was sent by a karate instructor from Tupelo, Mississippi, and the second set from a part-time actress and housewife from Dallas, Texas, who pleaded guilty to sending the letters in December 2013. These recent incidents demonstrated that a relatively low level of sophistication and technological knowledge were no bar to deployment of a WMD.
The ability of non-scientists to create and deploy a biological weapon highlights the emergence of a new threat, the “biohacker.” “Biohacking” is not necessarily malicious and could be as innocent as a beer enthusiast altering yeast to create a better brew. Yet the same technology used by a benign biohacker could easily be transformed into a tool for the disgruntled and disenfranchised to modify existing or emerging biological warfare agents and employ them as bioterrorism. A 2005 Washington Post article by Steve Coll and Susan Glasser presciently stated that “one can find on the web how to inject animals, like rats, with pneumonic plague and how to extract microbes from infected blood…and how to dry them so that they can be used with an aerosol delivery system, and thus how to make a biological weapon. If this information is readily available to all, is it possible to keep a determined terrorist from getting his hands on it?”
This article argues that the biohacker is a real and existing threat by examining evasive biohacking strategies and limitations of current detection methods. The article finds that more active measures are required to stem the growing, long-term threat of modified BW agents employed by individuals. The biohacker is not only a credible threat, but also one that can be checked through improved detection and by disrupting BW agent delivery methods.
The Danger of Biological Warfare Agents
Biological agent weapons, unlike conventional weapons or other WMD, have the potential to create a runaway uncontrollable event. The damage of a bomb or artillery shell is constrained by the blast radius. The effects of chemical and nuclear WMD dissipate over time, albeit with a broad range of half-lives, environmental diffusivities, and ease of decontamination. In contrast, BW are microorganisms that upon dissemination could proliferate exponentially within a single host, linger, and spread from one host to another. BW, therefore, have the potential to be unbounded in both space and time. The hosts themselves serve as potent amplifiers for the agent. Common to all BW agents is the existence of a lag time between time of infection and onset of symptoms. This lag time or incubation period allows infected individuals to feel healthy and to continue with their lives asymptomatically, which increases the potential for spreading.
The Defense Advanced Research Projects Agency (DARPA) commissioned a JASON study in 2003 to examine the best means to detect, identify, and mitigate the effects of a biological agent release within the United States. The study emphasized that current technologies and those expected to be developed within the next five years could not achieve a nationwide blanket of biosensors. Instead, sensors that are currently available should be used at critical locations according to a pre-established “playbook.” Outside the range of these critical nodes, biosurveillance against a bioterrorism event would be accomplished through medical surveillance. The essential component of such surveillance would be the “American people as a network of 288 million mobile sensors with the capacity to self-report exposures of medical consequence to a broad range of pathogens.” As a result of the H1N1 flu pandemic, the 2012 National Strategy for Biosurveillance further reiterated the findings of the JASON report and called for medical biosurveillance to move beyond chemical, biological, radiological and nuclear (CBRN) threats. This expansion increases medical surveillance to examine a “broader range of human, animal, and plant health challenges,” in an effort to improve early detection of emerging diseases, pandemics, and other exposures.
Medical biosurveillance, however, has an intrinsic limitation: it is entirely dependent on the self-reporting of symptoms and illnesses, which only occurs after an incubation period. This time lag is the window of opportunity for malicious activity by the biohacker aimed at increasing the damage and spread of BW effects. For example, delayed onset of symptoms and ease of international travel enable an individual from the United States to be anywhere in the world within a few hours of BW exposure, potentially infecting hundreds if not thousands along the way. From the biohacker’s point of view, a highly virulent pathogen with a short incubation interval and rapid mortality may not be as desirable as a less virulent one, which would allow the infected individuals to travel greater distances before exhibiting symptoms or dying. A biohacker possesses several strategies to maximize the BW incubation period to evade or alter the medical biosurveillance network.
Strategies of the Biohacker
Many biological warfare agents are naturally occurring around the world or easily derived from plants and could be transformed by biohacking. The advent of modern technologies enables the biohacker to employ one or a multitude of strategies to increase the tactical or strategic effectiveness of a biological agent. The authors distinguish five of these strategies as “Wolf in Sheep’s Clothing,” “Trojan Horse,” “Spoof,” “Fake Left,” and “Roid Rage.”
A “Wolf in Sheep’s Clothing” occurs when a biological organism or toxin is modified through genetic engineering so that it can be expressed in an active form but does not present the normal epitopes. In a “Trojan Horse,” a biohacker maintains the epitope of a non-threatening agent but re-engineers the active component of the toxin to increase the biological threat without increasing the detectability. The “Spoof” occurs when a benign agent is modified to express epitopes distinctive of a known toxin in order to trigger an unnecessary protective response by the target parties (the local, state, or federal government), while the delivering party (the biohacker) can afford to remain unencumbered. The “Fake Left” is a means to modify through selection or genetic engineering the method of transmission of an organism (for example, one that is typically passed by fluid to an airborne method). Such modification makes it easier to disperse an agent among a target population. The “Roid Rage” strategy aims to potentiate the effects of a common virus by expressing components of a deadly virus, such as expressing Ebola virus RNA sequences into the common flu virus. An infected person would demonstrate symptoms of the flu, hampering early detection and treatment of Ebola and favoring its deadly outcomes.
Any of these strategies could be used separately or in conjunction with one another. These strategies also do not require large or sophisticated laboratories to accomplish. Moreover, at the biohacker’s disposal is a plethora of scientific data. For example, an article from a major medical journal published last year on the avian flu virus highlighted the five specific genetic modifications required to transmit the virus from ferret to ferret, a model used since ferrets are susceptible to the same flu viruses as humans. Such information provides a framework for biohackers to implement their strategy.
Defending Against the Biohacker
Improving Detection Methods
Advances in biotechnology and genetic engineering facilitate the modification of more BW agents with increased toxicity, transmissibility, and lethality. Many bioengineering companies around the world now openly sell “all-in-one” kits for researchers to perform recombinant DNA experiments. Such kits are available to the public and provide the ability to modify known bioagents. Technological advances and lowering costs make the biohacker a viable threat, but they also enable counter-bioterrorism through cheaper and more reliable detection and identification systems.
One accessible means to thwart the biohacker is the development of physical, chemical, and biological sensors that reliably detect and identify a biological agent by its mechanism of action. For example, the International Genetically Engineered Machine (iGEM) Foundation supports a yearly competition in which competitors are given a “kit of biological parts” and through their own design are expected to build synthetic “biological systems and operate them in living cells.” The innovative goals of the iGEM competition in its 242 laboratories worldwide are to promote biosafety and biosecurity by focusing on therapeutics or toxin detection/identification.
Detecting Host Response
Of the various strategies to detect a toxin, the most straightforward focuses on the specific molecular epitope of the active agent, either through molecular recognition (for example, a distinctive surface protein on the organism), or the detection of genetic material specific to a particular pathogen. Unfortunately, these signals can be very weak early into the infection of an individual, and the organisms themselves may be sequestered from ready observation, as was the case with the AIDS virus. The solution to these problems is to continue to increase the sensitivity and specificity of the detection methods, but this in turn may increase vulnerability to hacking. Since these methods depend heavily on the ability to detect specific epitopes, several of the biohacking strategies listed above could be utilized. An alternative approach is to focus not on the agent itself, but on the host response to the agent. In this case, the host serves as an amplifier that produces a multitude of cellular signaling molecules that can potentially be measured to provide an identifying signature, ideally before the onset of clinical symptoms. The host response does not need to be measured in a person since live cell bioreactors with orthogonal quantitative measurements could provide the identifiable signature. While there is no guarantee that the detailed dynamic host response will be pathogen-specific, early detection of an infection is still beneficial by triggering the administration of a drug, a cytokine, or a combination thereof to block progression of the infection.
One intrinsic limitation of biohacking is the delivery system. Microorganisms require either a host or stable laboratory cell culture conditions to survive, but some can be effectively placed in a passive spore state that simplifies transmission. Delivery of biological agents is not trivial. Many biological agents, such as anthrax and ricin, are not transmittable from person to person, hence the delivery of millions of spores over a large area is required. Yet, conventional dispersion through munitions would destroy the spores or toxin. The “weaponization” of pathogens may require a certain level of sophistication, but even a non-weaponized agent can have a significant psychological effect. Agents such as Ebola, which are transmittable from person to person, are relatively unstable outside the host, further complicating delivery. Smallpox is an example of a potential agent that is transmittable from person to person and is stable outside the host, but the potential for infection is limited by the smallpox vaccine. Programs to develop specific vaccines, particularly those for animal-borne disease, could provide additional protection. Through knowledge management of scientific data, it might be possible to impede the development of a stable delivery system for malicious purposes.
In addition, certain equipment and materials, such as fermenters, incubators, enzymes, and retroviruses, are required to modify agents. Limiting the sale or, at the least, monitoring the sale of such materials would also make it difficult for a biohacker to create a modified biological agent undetected. Some of the technologies are simple enough, however, that they could be adapted from readily available consumer items, and even the more complex biological reagents, such as lenti-viruses and specific cell lines, are readily obtainable through research supply companies. Given these challenges, it is important to maintain a strong national effort in detection and prophylaxis bioagent production equipment and supplies.
Unlike conventional weapons or other WMD forms, biological weapons are difficult to contain. The time period that naturally occurs between release and identification provides an opportunity for the pathogen to spread silently. This time period could increase if the biohacker becomes more skilled at hiding agents or modifying incubation times, causing increased transmission. Current detection methods, such as medical biosurveillance and the Joint Biological Point Detection System, abide by the detect-to-treat mentality: they are passive and geared to react to signs of an outbreak or bioagent deployment. In contrast, modern technology makes it possible to move to a detect-to-prevent strategy. The key to such a strategic leap is to reduce drastically the lag time required to correctly identify the biothreat and respond accordingly.
The paradox of new scientific methods and technology, however, is that they lead not only to new discoveries in terms of medicine, but also provide information that enables the biohacker. In a world accustomed to well-defined toxin epitopes and detector receiver-operator characteristics, the hacking of a toxin can be manifested in many ways; for example, in the presentation of an unexpected epitope that could render an existing detection platform ineffective. The modification of BW agents not only makes their identification difficult, but also may render the known therapeutic methods ineffective.
The 2013 ricin letters, in conjunction with the multitude of low-cost tools and strategies available, highlight that the biohacker is a real and contemporary threat. Combating the capabilities of the biohacker will be neither easy nor inexpensive. Although the biohacker still has significant obstacles of production and dispersion to overcome to effectively devastate a large population, the availability of technology and scientific information makes this an impending danger. Continued research is required to develop identification tools that are in front of the medical biosurveillance lag time. The fiscal costs of biodefense are high for continued research and development, but the risk of not stemming the means of the biohacker is even greater.
Captain Stephen Hummel is an FA52 officer who is currently studying Chemical and Physical Biology at Vanderbilt University as part of the Army’s Advance Civil Schooling Program. CPT Hummel previously served in both Iraq and Afghanistan and as the USAREUR CBRN plans officer. Upon graduation from Vanderbilt, CPT Hummel will teach in the Chemistry and Life Science Department at the U.S. Military Academy, West Point.
Vito Quaranta, MD, is a Professor of Cancer Biology, Department of Cancer Biology, Vanderbilt University Medical School, Vanderbilt University, and the Director of the Center for Cancer Systems Biology@Vanderbilt, funded by the National Cancer Institute. He conducts a systems-informed effort to characterize the dynamics of cellular responses to perturbations in the context of anticancer drug discovery.
John P. Wikswo, Ph.D., is the Gordon A. Cain University Professor, A. B. Learned Professor of Living State Physics, and Professor of Biomedical Engineering, Molecular Physiology and Biophysics, and Physics at Vanderbilt University. He is also the founding Director of the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE), where he is directing a large effort in the development of microfabricated devices and organs-on-chips for systems biology, drug development/toxicology/safety, and biodefense.
Acknowledgement: The preparation of this manuscript was funded in part by Defense Threat Reduction Agency grants HDTRA1-09-1-00-13 and DTRA100271A-5196 and DARPA grant W911NF-12-2-0036. The viewpoints expressed in this article do not necessarily reflect those of the U.S. Army or the Department of Defense. The authors thank David Cliffel and John McLean for their comments and suggestions, and Allison Price for her editorial assistance.
 Friedrich Frischkneckt, “The History of Biological Warfare: Human Experimentation, Modern Nightmares and Lone Madmen in the Twentieth Century,” EMBO Reports 4:S47-S52 (2003).
 “The Biological Weapons Convention,” United Nations Office for Disarmament Affairs, available at www.un.org/disarmament/WMD/Bio/.
 There is a distinction between “offensive” and “defensive” biological warfare agents. Signatory countries are allowed to maintain secure stockpiles of biological agents to maintain vaccine and antidote stores.
 The list of non-signatory states can be found at www.opbw.org.
 Albeit in smaller quantities than could be produced by a nation-state.
 The Centers for Disease Control and Prevention defines bioterrorism as “the deliberate release of viruses, bacteria, or other germs (agents) used to cause illness or death in people, animals, or plants.” See the Centers for Disease Control and Prevention, available at www.emergency.cdc.gov/bioterrorism/overview.asp.
 On February 19, 2010, the U.S. Justice Department, the FBI, and the U.S. Postal Inspection Service formally concluded the investigation into the 2001 anthrax attacks and issued an Investigation Summary. Dr. Ivins took his own life before charges could be filed against him.
 The term “support” means both physical and financial support; specifically, access to laboratories such as research universities or pharmaceutical companies.
 “Trial of Mississippi Man Charged with Sending Ricin Letters May be Delayed,” Associated Press, September 13, 2013.
 Shannon Richardson pleaded guilty on December 10, 2013, to sending the ricin-laced letters in an effort to frame her husband. See “Texas Woman Pleads Guilty to Sending Ricin to President,” Associated Press, December 11, 2013.
 Ricin is a small, toxic carbohydrate-binding protein found in castor oil beans. To be an effective BW agent, it must be extracted from the beans and purified to a concentration to deliver an aerosolized dose of a specific range of micrograms per kilogram. Consequently, for a person weighing 180 pounds, a specific amount of micrograms of ricin would need to be present for it to be fatal. (In terms of sophistication, a ricin letter is a simple device and does not require a complex dispersion method since it is presumed that the person opening the letter is the intended target. Neither the exact concentration nor dispersal properties of the ricin in the letters have been made public; however, the concentration was high enough to set off detectors in the mail-processing facilities.) For more details on ricin, see “Biosafety in Microbiological and Biomedical Laboratories,” Centers for Disease Control and Prevention, December 2009.
 A bioterrorist is one who simply employs biological weapon agents unmodified, while a terrorist biohacker is one who modifies a known toxin or biological agent with malicious intent.
 Steve Coll and Susan B. Glasser, “Terrorists Turn to the Web as Base of Operations,” Washington Post, August 7, 2005.
 The impact of BW agents on human health proceeds from organ failure and tissue destruction, but is ultimately defined by toxic effects on cellular functions, with the most severe being cell death. Consequently, before the effects are seen at the level of the organism, they occur on the molecular and cellular scale, and continue from the point of infection and even beyond the appearance of medical symptoms. Presymptomatic detection of early signatures of an infection could mitigate some threats.
 The study was conducted by JASON, an independent group of scientists operating through the MITRE Corporation, who advise the U.S. government on issues related to science and technology.
 “Biodetection Architectures,” JASON, February 2003.
 The Joint Biological Point Detection System (JBPDS), a continuous environmental aerosol monitor, is currently available for point biosurveillance. These devices collect samples over a certain interval and then the sample is transported to a “central laboratory” for analysis. Detection of a biological agent in a city, for example, would require a large area of systems and technicians not only to collect samples but also to test them. The cost of extending the JBPDS beyond the few existing, strategic locations would be overwhelming. Advances in technology will undoubtedly produce compact, lower-cost automated detection systems that could be much more widely disseminated, but this then presents an increased risk for accidental or intentional false alarms and hence requires a rapid and highly accurate second-level validation.
 The value of 288 million is based on the census data available during the 2003 study by JASON.
 “Biodetection Architectures.”
 The National Strategy for Biosurveillance signed in July 2012 specifically said, “Where efforts since the tragic terrorist attacks of September 11, 2001, have focused largely on threats associated with the deliberate use of CBRN weapons, this Strategy embraces the need to engage in surveillance for WMD threats and a broader range of human, animal, and plant health challenges, including emerging infectious diseases, pandemics, agricultural threats, and food-borne illnesses.”
 For example, ricin can be derived from castor oil beans, anthrax can be found in the soil around certain domestic and wild animals, botulism, which is endemic in some environments, can be cultured by anaerobic purification of meat, and plague can be carried by wild rodents and transmitted to humans via fleas.
 This classification of biohacker strategies was developed by John Wikswo, David Cliffel, and John McLean at Vanderbilt University and presented in December 2012 at the Johns Hopkins University Applied Physics Laboratory’s Cellular Sensing Systems Workshop.
 Epitopes are specific amino acid sequences on the surface of a cell, or certain BW agents such as anthrax that invoke a specific immune response. The unique amino acid sequences are identifiable traits of certain BW agents and are viewed as biomarkers. The concept of epitope can be extended to include any amino acid sequence that can be detected though a molecular affinity assay, such as aptamers. See, for example, Larry Gold et al., “Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery,” PLOS One 5:12 (2010). Separately, gene expression dynamic inspection (GEDI) studies (Sui Huang et al., “Cell Fates as High-Dimensional Attractor States of a Complex Gene Regulatory Network,” Physical Review Letters 94 (2005)) demonstrate that HL-60 under different environmental conditions will present different genes throughout their transformation process to neutrophils, 168 hours later. So identification through gene expression at a given time point would identify two different agents. The concept of gene expression phase space and epigenetic attractors is treated in more detail in Sui Huang and D. E. Ingber, “A Non-Genetic Basis for Cancer Progression and Metastasis: Self-Organizing Attractors in Cell Regulatory Networks,” Breast Disease 26 (2007).
 The Aum Shinrikyo cult in 1993 twice dispersed large amounts of anthrax around Tokyo using a variety of methods. The anthrax strain acquired by the cult was designed as a vaccine for cattle, and therefore did not have any effect on humans. See Amanda Onion, “Lessons from Failed 1993 Biological Attack,” ABC, October 5, 2001.
 One such example is the “AllPrep DNA/RNA/Protein Mini Kit” by Qiagen Technologies. A single kit, which can be used on 50 samples, costs $565.
 Recently published articles highlight that the presence of biological weapons agents can be measured in real time and at minimal concentrations. Work by Eklund et al. (2009) demonstrated the dynamic metabolic responses in microphysiometer experiments to 100 nano-molar ricin and 1 to 2 micro-molar anthrax concentrations.
 See the International Genetically Engineered Machine (iGEM) Foundation, located at www.igem.org.
 J. Gallarda et al., “Early Detection of Antibody to Human Immunodeficiency Virus Type 1 by Using an Antigen Conjugate Immunoassay Correlates with the Presence of Immunogloblin M Antibody,” Journal of Clinical Microbiology 30:9 (1992).
 A recent study examined the blood transcriptome of volunteers inoculated with influenza and developed a predictive signature. See Christopher Woods et al., “A Host Transcriptional Signature for Presymptomatic Detection of Infection in Humans Exposed to Influenza H1N1 or H3N2,” PLOS One 8:1 (2013).
 This means that by using a variety of instruments and technology in a cellular culture system, it is possible to measure changes in proteins, electro-chemical responses, changes in cellular morphology and changes in cell motility. These simultaneous measurements provide a unique signature which is distinct for each bio-agent, making early detection possible by detecting the initial cellular changes.
 See X. Qui et al., “mAbs and Ad-Vectored IFN-alpha Therapy Rescue Ebola-Infected Nonhuman Primates When Administered After Detection of Viremia and Symptoms,” Science Translational Medicine 5:207 (2013): pp. 143-153, for a recent report on blocking Ebola in nonhuman primates by using a combination of antibody therapy cocktails and interferon.
 Harvey Lodish et al., Growth of Microorganisms in Culture, in Molecular Cell Biology (New York: W.H. Freeman, 2000); V. R. Dowell, Jr., et al., “Coproexamination for Botulinal Toxin and Clostridium Botulinum. A New Procedure for Laboratory Diagnosis of Botulism,” Journal of the American Medical Association 238:17 (1977); J.R. Koransky, S. D. Allen and V. R. Dowell, Jr., “Use of Ethanol for Selective Isolation of Sporeforming Microorganisms,” Applied and Environmental Microbiology 35:4 (1978).
 “FM 3-11.9 Potential Military Chemical / Biological Agents and Compounds,” U.S. Army Chemical School, January 2005.
 “A World Free of One of the Most Virulent Animal Diseases?” U.S. Department of Homeland Security, undated.
 There are several websites through which biohackers share information. A few that are easily found are DIYbio.com, grindhousewetware.com, and dangerousthings.com. If not already in place, a rigorous program to monitor such sites should provide valuable intelligence on possible emerging threats and low-cost BW technologies.
 The FY2013 Federal Budget allocated for biodefense was $5.53 billion including monies for basic research for vaccines as well as advanced research for detection. See C. Franco and T. Sell, “Federal Agency Biodefense Funding, FY2012-2FY013,” Biosecurity and Bioterrorism 10:2 (2012).