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Biological agents: Weapons of warfare and bioterrorism
Molecular Diagnosis Vol. 6 No. 4 2001
Biological Agents: Weapons of Warfare and Bioterrorism LARRY A. BROUSSARD, PhD New Orleans, Louisiana
The use of microorganisms as agents of biological warfare is considered inevitable for several reasons, including ease of production and dispersion, delayed onset, ability to cause high rates of morbidity and mortality, and difficulty in diagnosis. Biological agents that have been identified as posing the greatest threat are variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filoviruses (Ebola hemorrrhagic fever and Marburg hemorrhagic fever), and arenaviruses Lassa (Lassa fever) and Junin (Argentine hemorrhagic fever). The pathogenesis, clinical manifestations, diagnosis, and treatment of these agents are discussed. Rapid identification and diagnosis using molecular diagnostic techniques such as PCR is an essential element in the establishment of coordinated laboratory response systems and is the focus of current research and development. Molecular techniques for detection and identification of these organisms are reviewed. Key words: biosensors, anthrax, smallpox, hemorrhagic fever.
increased the fear that the weapons and agents developed may be obtained by, or the personnel involved in these programs may be recruited by, other nations or terrorist groups. The use of Salmonella typhimurium by the Rajneeshee cult in 1984 to intentionally contaminate salad bars in Oregon restaurants [3] and the release of the nerve gas sarin in a Tokyo subway in 1995 by the Aum Shinrikyo cult [4] illustrate the feasibility of use of such agents by groups for political, religious, or other purposes. Recent epidemics of “mad cow” and foot-and-mouth diseases illustrate the potential devastation of antiagriculture biological agents. Depending on the intent of the person, group, or nation using biological agents, the incident may be overt (announced) or covert (unannounced). In either case, if the target is civilian the clinical molecular microbiologist will play a key role in the detection and identification of the agent. For covert incidents, the clinical microbiology laboratory may not only make the initial identification of the organism but also be responsible for recognizing
The terrorist attacks of September 11, 2001 and the subsequent incidents involving anthrax have increased concern about the use of chemical or biological agents by countries, terrorist groups, or individuals. The existence of offensive biological warfare programs in the former Soviet Union and Iraq [1] shows the willingness of some nations to continue such programs even after signing the 1972 Convention on the Prohibition of the Development, Production, and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction (BWC) [2]. The breakup of the Soviet Union and its subsequent economic problems have From the Department of Clinical Laboratory Sciences, Louisiana State University Health Sciences Center, New Orleans, LA. Reprint requests: Larry A. Broussard, PhD, Department of Clinical Laboratory Sciences, Louisiana State University Health Sciences Center, 1900 Gravier St, New Orleans, LA 70112. Email: [email protected] Copyright © 2001 by Churchill Livingstone威 1084-8592/01/0604-0013$35.00/0 doi:10.1054/modi.2001.29155
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the outbreak as an incident of bioterrorism based on the pattern and frequency of symptoms and positive test results. The clinical molecular microbiologist must be familiar with the characteristics of these agents and the technologies available for their detection and identification. If the laboratory does not have the capability to handle and detect these agents, personnel must be familiar with proper handling techniques and have protocols in place for sending the samples to the nearest laboratory with the required capability. The chemical and biological agents that pose the greatest threat to civilian and military populations have been reviewed by several authors [5–9]. Biological warfare agents are easy to manufacture, generally don’t require sophisticated delivery systems, have the ability to kill or injure large populations, cause illnesses that are unrecognized in their initial stages, and may affect large geographical areas if asymptomatic infected individuals travel [5]. Since the termination of the United States offensive biological weapons program by executive orders of President Nixon in 1969 and 1970, many U.S. government agencies have conducted research and developed programs for the detection and identification of biological agents and the prevention and treatment of illnesses caused by these agents. The Centers for Disease Control and Prevention (CDC) Strategic Planning Workgroup recommends that preparedness efforts focus on agents with the potential for the greatest impact on health and security, especially those that are highly contagious or that can be engineered for widespread dissemination via aerosols and have designated these agents as category A [6]. Category A agents include Bacillus anthracis (anthrax), variola major (smallpox), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filoviruses (Ebola and Marburg hemorrhagic fevers), and arenaviruses (Lassa-Lassa fever, Junin-Argentine hemorrhagic fever, and related viruses). The “high priority” biological agents listed in the North Atlantic Treaty Organization (NATO) Handbook on Biological Terrorism include five of the same agents but substitute Brucella species (brucellosis) for the hemorrhagic fever viruses [8]. This article includes a review of the category A agents, including some of the molecular techniques associated with their detection and identification and the development of protective measures against them. These agents include bacteria, a toxin produced by bacteria, a DNA virus, and
RNA viruses. Because similar molecular techniques are often adopted for detection of organisms within the same class (bacteria, RNA virus, DNA virus), each technique is discussed at its first mention. Most of the current research and development is either government funded or performed by the military because of biowarfare implications, but the nonmilitary clinical molecular microbiologist should remain knowledgeable about current developments because of potential terrorist incidents.
Anthrax B. anthracis, an aerobic spore-forming, grampositive rod, causes anthrax (from the Greek for coal because of the black, coal-like cutaneous lesions of the disease). Anthrax spores are very resistant and may remain dormant in the soil for decades. In 1970, a report of the World Health Organization (WHO) estimated that the release of 50 kg of anthrax spores along a 2-km line upwind of a city of 500,000 would result in 95,000 deaths [10], and a 1993 report of the U.S. Congressional Office of Technology Assessment predicted that release of 100 kg of anthrax spores upwind of Washington, DC, would result in 130,000 to 3 million deaths [11]. Germany used B. anthracis to infect livestock and contaminate animal feed to be exported during World War I, Japan used it in World War II, and the organism was included in the programs of the Soviet Union, Iraq, and the United States [2]. The lethal potential of aerosolized anthrax spores was demonstrated by 68 deaths following their accidental release in 1979 from a military facility in Svedlovsk in the former Soviet Union [12]. In humans, anthrax has 3 clinical presentations: cutaneous, gastrointestinal, and inhalational. Cutaneous anthrax, which typically follows exposure to anthrax-infected animals, is the most common naturally occurring form. Gastrointestinal anthrax, which follows ingestion of insufficiently cooked contaminated meats, is fairly uncommon [13]. Inhalational anthrax, known as woolsorters’ disease, was an occupational hazard of workers in slaughterhouses and textile and tanning industries handling contaminated wool, hair, meat, and hides but has been almost completely eliminated in Western nations by immunization. Inhalational anthrax is the form most likely to result from a biological attack. Anthrax spores have a
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diameter of 2 to 6 µm, which is ideal for impinging on human lower respiratory mucosa. After inhalation, spores are phagocytized by tissue macrophages and then germinate into bacilli, which produce a necrotizing hemorrhagic mediastinitis [7]. The time for infection is variable and in Sverdlovsk cases occurred from 2 to 43 days after exposure. Once germination occurs, the disease progresses rapidly. There is no evidence that an immune response is initiated against vegetative bacilli. Anthrax bacilli release two toxins (edema and lethal) leading to hemorrhage, edema, necrosis, and systemic effects that lead to death. Edema toxin consists of protective antigen (PA), which permits entry of the toxin into the host cell, and edema factor, a calmodulin-dependent adenylate cyclase that increases intracellular levels of cyclic adenosine monophosphate, upsetting water homeostasis. Lethal toxin consists of PA and lethal factor, a zinc metalloprotease that inactivates mitogen-activated protein kinase in vitro and stimulates macrophages to release tumor necrosis factor ␣ and interleukin 1 [14]. The exotoxins are thought to inhibit the immune response. The three exotoxin components (PA, lethal factor, and edema factor) are encoded on virulence plasmid pXO1, which is 185.5 kilobase pairs (kbp) in size. The other virulence plasmid, pXO2, is 95.3 kbp and codes for 3 genes (capA, capB, and capC) associated with the synthesis of the polyglutamyl capsule, which inhibits phagocytosis of vegetative anthrax bacilli. Host-specific factors such as temperatures greater than 37°C, CO2 concentration greater than 5%, and the presence of serum components regulate the expression of virulence factors. The transcriptional activator AtxA mediates the regulation of the toxin and capsule genes. The transcriptional activator AcpA also controls expression of the capsule gene. Full virulence requires expression of both plasmids. The Pasteur strain carries pXO2 and does not express exotoxin components; the Sterne strain carries pXO1 but does not have a capsule [14]. The vaccine developed in England is an alumprecipitated cell-free filtrate of a Sterne strain culture and induces measurable antibodies against lethal and edema factors but low levels of antibody to protective antigen [15]. The only licensed vaccine (Bioport Corporation, Lansing, MI) currently available in the United States is an alum-absorbed, partially purified culture filtrate of the avirulent, nonencapsulated strain B. anthracis V770-NP1-R
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[16]. It has been shown to induce high levels of antibody only to PA and requires injections at 0, 2, 4, 6, 12, and 18 months and annually thereafter. Presently all of the available supply is being used for immunization of military personnel. This vaccine is not highly purified and contains multiple extraneous proteins. Current research is focused on developing vaccines with a simpler schedule and better protection. Vaccines now being tested include preparations of PA subunits with different adjuvants, PA purified from recombinant sources, and live vaccines based on anthrax strains with auxotrophic mutations [14]. A vaccine based on purified PA made by recombinant technology has been protective in animals [17]. Inhalational anthrax has initial symptoms (fever, nonproductive cough, malaise, fatigue, and myalgia) similar to those of a viral respiratory infection and is essentially impossible to diagnose in the absence of a known outbreak. Respiratory distress ensues, with shock and death following in less than 24 hours. A widened mediastinum on chest x-ray findings in a previously healthy patient with evidence of overwhelming flulike illness is essentially pathognomonic of advanced inhalational anthrax. Recommended treatment is ciprofloxacin or doxycycline as initial therapy, with penicillin as an alternative once sensitivity data are available [18]. The possibility of genetically engineered resistant strains exists, and there has been one report that Russian scientists developed a strain resistant to tetracycline and penicillin class antibiotics [19]. Laboratory diagnosis of anthrax involves identification of B. anthracis microbiologically, serologically, or by use of more sensitive molecular techniques. B. anthracis forms rough gray-white colonies with characteristic “comet-tail” protrusions on sheep blood agar [15]. It is aerobic, Grampositive, spore-forming, and nonmotile. Polymyxinlysozyme-EDTA-thallous acetate agar is used as a selective medium for B. anthracis if samples are likely to be contaminated with other bacillus species [20]. Although growth should occur within 24 hours, identification of a Bacillus species from a blood culture may require another 24 hours, and the laboratory may not further identify the Bacillus species unless specifically requested to do so [13]. Immunologic tests include specific enzyme-linked immunosorbent assays (ELISAs) to measure antibody titers to PA or to capsular components. Indirect microhemagglutination gives similar results to ELISA but
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has drawbacks, including short shelf life of reagents and longer preparation times [14]. Among the most sensitive methods are the immunomagneticelectrochemiluminescence (ECL) assays for antigen detection and PCR gene amplification for nucleic acid detection [21]. Automated applications are being developed with the ultimate goal of developing portable, hand-held devices capable of rapid detection of all biological threat agents. The ECL system is described in detail by Higgins et al. [21]. Magnetic beads conjugated to a capture antibody and a ruthenium-conjugated detector antibody are used to achieve sensitivities in the range of 0.1 to 1.0 pg/mL. Chemiluminescence is generated by a voltage-dependent reaction of ruthenium heavy metal chelate. This technology is adaptable for use in detection of any biological agent, and its primary limitation is dependence on the availability of high-quality antibodies and other ligands. Detection of protein toxins, not amenable to nucleic acid– based assays, is possible using ECL. Automated ECL detection systems have been developed with the capability of detecting several organisms, including B. anthracis PA antigen. Higgins et al. [21] compared several PCR-based techniques for the detection of pathogenic organisms. It is more difficult to extract DNA from B. anthracis spores than from vegetative cells. Germination of spores for 60 minutes before DNA extraction increased the sensitivity to that achieved with vegetative cells. Among the techniques evaluated for DNA extraction were the Qiagen, Inc (Valencia, CA) kits designed for tissue and blood using protease digestion and chaotropic salt–mediated lysis of cells and the Autolyser (XOHOX Research Institute, Menlo Park, CA) fully automated procedure. The Qiagen system was more sensitive for detection of B. anthracis, but the ease of use of the Autolyser justified further research. Preparation of B. anthracis DNA using FTA paper (Flinders Technologies, Inc, Adelaide, South Australia) and IsoCode cards (Schleicher and Schuell, Keene, NH) was also evaluated. The FTA procedure required several wash steps, compared with the single wash step for the IsoCode paper, and the IsoCode paper was more efficient for plasma or serum matrices. It was determined that the IsoCode extraction could be used to detect B. anthracis in milk at concentrations that may be encountered in a possible bioterrorism scenario [21]. Another technique that has been used for the identification of B. anthracis is the commercial
TaqMan (Perkin-Elmer/Applied Biosystems, Foster City, CA) 5' nuclease assay, which uses fluorogenic probe-based PCR [21]. This technique offers advantages of real-time monitoring, simultaneous assay for different target sequences through use of different oligonucleotide probes labeled with different dye molecules, and the ability to design probes to discriminate between single nucleotide differences in gene sequences. Using this technique, the Special Pathogens Department at the Diagnostic Systems Division of the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) analyzed material from an incident involving a preparation suspected of containing B. anthracis [21]. Assay time was approximately 2 hours. Positive results were obtained when the samples were analyzed with primers and probes for PA gene, and negative results were obtained when the samples were analyzed with primers and probes for capsular antigen gene. The lack of capsular antigen gene, which is associated with virulent strains of B. anthracis, and other data led the investigators to conclude that the sample was an avirulent strain conventionally used in vaccine preparation. Development of portable or handheld instruments for monitoring reactions of fluorogenic probe assays is an ongoing project. The Smartcycler (Cepheid, Sunnyvale, CA) is a commercially available real-time, fluorogenic probe–based PCR portable analyzer. The company plans to develop briefcase-size units [21]. The U.S. Department of Energy’s Lawrence Livermore National Laboratory developed and manufactured a suitcase-size device, MATCI (miniature analytical thermal cycler instrument) that has been used to detect and differentiate between orthopox virus species [22]. The next version of this biodetector was the ANAA (advanced nucleic acid analyzer), which consisted of an array of 10 silicon PCR reaction modules, each containing a silicon PCR microchip, a thermistor to interface the thermal controller with the microchip, and a complete set of optics for fluorogenic detection [23]. This instrument was used to analyze samples of several organisms including Bacillus subtilis spores (to simulate B. anthracis spores) and detection limits of 105 to 107 organisms/L were observed with detection times as short as 16 minutes. The most recent version of the instrument is the HANAA (handheld advanced nucleic acid analyzer), which was introduced in November 1999 and began beta testing by FDA inspectors in July
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2000 [24]. The instrument has four automatically calibrated sample modules and has typical detection times of 6 to 10 minutes [25].
Smallpox Smallpox, a viral disease that once occurred worldwide, exists in two principal forms: variola major and a much milder form, variola minor. Differentiation of the causative viruses is now possible. In 1980, the WHO declared smallpox eradicated after the last natural case of variola occurred in Somalia in 1977 [26]. Following a WHO recommendation that all laboratories destroy their stocks of variola virus or transfer them to one of two WHO-approved repositories, all countries reported compliance. The repositories are the CDC in Atlanta, GA, and the Institute of Virus Preparations in Koltsovo, Novosibirsk region, Russia. An expert committee of the WHO recommended that all viral stocks be destroyed by June 1999, but this action was delayed by another committee’s recommendation in December 1999 that further limited research on variola viruses could be justified but should not extend beyond the end of 2002. The existence of clandestine repositories has been rumored, and a former deputy director of the Soviet Union’s civilian bioweapons program, Ken Alibek, has reported that the Soviet Union has an industrial production capacity of tons of smallpox virus annually and even now has a research program seeking to produce more virulent and contagious recombinant strains [27]. The discontinuation of routine vaccination, high mortality rates (30% or higher), aerosol infectivity, and stability of the virus make it a very dangerous biological weapon threat. Smallpox (variola major), a DNA virus, is a member of the genus orthopoxvirus, which also includes monkeypox, cowpox, and vaccinia. The viruses are complex, and the virion is brick-shaped with a diameter of approximately 200 nm. Spread from person to person occurs via aerosols expelled from the ororpharynx of infected persons, by direct contact, or via contaminated clothing or bed linens. There are no known insect reservoirs or vectors. After aerosol exposure, the virus travels from the respiratory tract to the regional lymph nodes, where it replicates and gives rise to viremia on about the third or fourth day. After multiplication of virus in the spleen, bone marrow, and lymph nodes, a
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secondary viremia begins on approximately the eighth day and is followed by fever and toxemia. After the 12- to 14-day incubation period (range, 7 to 17 days) the patient experiences malaise, fever, rigors, vomiting, headache, and backache [28]. Two or three days later, a papular rash develops over the face and spreads to the extremities. Patients are infectious from the time of onset of the rash and should be isolated until all scabs separate [18]. The rash becomes vesicular, then pustular, and scabs begin to form on approximately the eighth or ninth day of rash. The scabs eventually separate, and pitted scarring develops. For fatal cases, death usually occurs during the second week. All smallpox lesions develop at the same pace, whereas with chickenpox, the disease most commonly confused with smallpox, scabs, vesicles, and pustules may be seen simultaneously on adjacent areas of skin. The chickenpox rash is more dense over the trunk (the reverse of smallpox), and chickenpox lesions are almost never found on the palms or soles [29]. Hemorrhagic and malignant smallpox are two other forms that are difficult to recognize. Hemorrhagic cases are uniformly fatal, and pregnant women appear to be unusually susceptible. The incubation time is somewhat shorter, and the illness is characterized by high fever and head, back, and abdominal pain. A dusky erythema develops, followed by petechiae and frank hemorrhages into the skin and mucous membranes, with death by the fifth or sixth day after onset of rash [28]. The symptoms are similar for the malignant form, which is frequently fatal. In this form, the lesions never progress to the pustular stage and gradually disappear without forming scabs if the patient survives [28]. Laboratory confirmation of a diagnosis of smallpox usually involves identification of the virus from vesicular or pustular fluid collected from scraped lesions using the blunt edge of a scalpel and a cotton swab. State or local health department laboratories should be contacted for shipping instructions, and only high-containment (BL-4) facilities should perform the testing [28]. Preliminary confirmation may include identification of the characteristic brick-shaped virions of orthopoxviruses using electron microscopy. Clinical presentation and medical history help identify cowpox and vaccinia, but differentiation between smallpox and monkeypox virions is virtually impossible without further testing. Ultimate identification and characterization of strains may be accomplished using PCR and
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restriction fragment length polymorphisms (RFLP) [30]. The gene for A-type inclusion body proteins for orthopoxvirus species has been used for the differentiation of these viruses using PCR [31,32]. Monkeypox and vaccinia virus DNAs were detected and differentiated on the basis of a singlebase polymorphism using a fluorogenic 5' nuclease PCR assay [33]. Primers targeting a DNA sequence of the orthopoxvirus hemagglutinin gene and two fluorescently labeled oligonucleotide probes were used in a single-tube assay. Ibrahim et al. [22] demonstrated detection of orthopoxviruses using the battery-powered MATCI. Consensus orthopoxvirus PCR primers were designed to amplify 266 to 281–bp segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus–specific fluorogenic (TaqMan) probe was designed and used to detect a single-base (A/C) substitution within the HA gene. Real-time detection was achieved in less than 1 hour. Currently there is no antiviral medication for use in infected patients. Cidofovir has been shown to have significant in vitro and in vivo activity against Poxviridae [7] and is being considered as a possible therapeutic agent for military personnel. Vaccination within the first 4 days after exposure may prevent or significantly ameliorate illness, and it has been recommended that all exposed persons as well as health care workers and disaster response personnel receive immediate vaccination in the event of an aerosol release of smallpox. A droplet of vaccine is administered in an area approximately 5 mm in diameter by rapid repeated (15 strokes) injection using a bifurcated needle. The site is covered to prevent touching or possibly transferring the virus to other parts of the body [28]. A vesicle typically appears in 5 to 7 days, forms a scab, and gradually heals within 2 weeks. Complications are the lowest of any vaccinia virus strain but may include postvaccinial encephalitis, progressive vaccinia, eczema vaccinatum, and generalized vaccinia [28]. Because of these complications, the Working Group on Civilian Biodefense recommended development of a vaccine from a more attenuated strain that would retain full efficacy [28]. Such a vaccine derived from a Lister strain was developed in Japan in the mid-1970s and given to more than 100,000 persons in Japan. Vaccinia immune globulin is indicated for treatment of complications of the smallpox vaccine. In the United States, approxi-
mately 140,000 vials of vaccine (50 to 60 doses/vial) are stored at the CDC, and it is estimated that 50 to 100 million doses are available worldwide. This vaccine, produced in the 1970s, consists of vaccine virus (New York Board of Health strain) grown on scarified calves [28]. In 2000, the CDC contracted Oravax of Cambridge, MA, to produce 40 million doses of smallpox vaccine with an anticipated delivery of the first production lots in 2004 [34]. The WHO Advisory Committee on Variola Virus Research agreed that determination of the fulllength genome sequences of additional strains might facilitate design of new drugs and better detection probes when it recommended further limited research on variola virus. Complete sequence information is currently available for two Asian variola major strains and alastrim (variola minor), and partial information is available for two African variola major strains, one variola minor strain, and the Butler and Harvey laboratory strains. Strains to be sequenced include Congo 70 (an African variola major strain) and Somalia 77 (a variola minor strain) [35]. The committee believed the use of variola virus for other research was not justified and suggested that use of alternatives such as surrogate orthopoxviruses was sufficient.
Plague Pandemics of plague, the disease caused by the bacteria Y. pestis, have occurred for centuries and caused the death of millions. During the pandemic beginning in 1346, plague, also known as the black death or great pestilence, killed 20 to 30 million Europeans [36]. Plague remains an enzootic infection of rats, ground squirrels, prairie dogs, and other rodents, and human plague occurs most commonly when plague-infected fleas bite humans, who then develop bubonic plague. Outbreaks of plague in China are attributed to plague-infected fleas dropped by a secret branch of the Japanese army, unit 731, during World War II [37]. The U.S. and Soviet biological weapons programs developed techniques to directly aerosolize plague particles that cause pneumonic plague, a highly lethal and potentially contagious form. An attack today would almost certainly occur via aerosol dissemination of Y. pestis, resulting in an outbreak of pneumonic plague.
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Pneumonic plague begins after an incubation period of one to six days, with high fever, chills, headache, and malaise followed by cough with hemoptysis, progressing rapidly to dyspnea, stridor, cyanosis, and death [18]. A 70-kbp virulence plasmid (pYV) enables Y. pestis to survive and multiply in the host. It encodes the Yop virulon, a system consisting of secreted proteins called Yops and their dedicated type III secretion apparatus called Ysc. This Yop virulon allows extracellular Y. pestis to inject the Yops, which block production of proinflammatory cytokines and induce apoptosis of macrophages [38]. Plague should be suspected whenever large numbers of previously healthy individuals develop fulminant Gram-negative pneumonia, especially if hemoptysis is present [18]. Y. pestis shows bipolar (safety pin) staining with Wright, Giemsa, or Wayson stain. It is a lactose nonfermenter, is urease and indole negative, and colonies are initially much smaller than other Enterobacteriaceae. Observable growth on blood or MacConkey agar typically takes 48 hours. Some automated bacterial identification systems may misidentify Y. pestis [39]. Most strains of Y. pestis produce an F1-antigen in vivo, which can be detected in serum samples by immunoassay [18]. A 5' nuclease PCR (TaqMan) assay targeting the plasminogen activator gene (pla) of Y. pestis was developed by Higgins et al. [40]. The assay detected the organism in experimentally infected fleas and monkey blood and oropharyngeal swabs. It has a detection threshold of 2.1 ⫻ 105 copies of the pla target or 1.6 pg of total cell DNA. The portable ANAA microchip PCR array instrument previously described detected Erwinia herbicola vegetative cells, the nonpathogenic simulator of Y. pestis, with a minimum detection limit of 105 cells/L [23]. Pneumonic plague is invariably fatal if antibiotic therapy is delayed more than 1 day after the onset of symptoms. Recommended antibiotic treatment is streptomycin, gentamicin, ciprofloxacin, or doxycycline administered for 10 to 14 days. Doxycycline is recommended for prophylactic treatment of asymptomatic exposed individuals, and alternative antibiotics include ciprofloxacin, tetracycline, and chloramphenicol [18]. No vaccine is currently available for prophylaxis of plague. The United States–licensed formaldehyde-killed whole bacilli vaccine discontinued in 1999 was effective against bubonic plague but not against pneumonic plague [37]. An
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F1–V antigen (fusion protein) vaccine protected mice for a year against an inhalational challenge and is now being tested in primates [18].
Botulism The anaerobic bacteria C. botulinum produce botulinum toxins, proteins of molecular weight approximately 150,000, which are the most toxic compounds known. There are 7 distinct neurotoxins, A through G, produced by different strains of the bacillus. Botulinum toxin is 100,000 times more toxic than sarin. As a biological weapon, botulinum toxins may be aerosolized or used to sabotage food supplies [7]. The botulinum toxins produce a blockade of acetylcholine release. They are zinc-dependent endopeptidases that cleave polypeptides essential for the docking of synaptic vesicles to the presynaptic membrane of the nerve terminal. Neurotoxins A and E target a protein of the presynaptic membrane, SNAP25, and neurotoxin B cleaves VAMP/synaptobrevin, a protein of the neurotransmitter-containing vesicles [41]. Inhalation of the toxins causes the same symptoms as ingestion, although the time course may be different. Onset of symptoms usually occurs 12 to 36 hours after exposure. Initial symptoms include cranial nerve palsies, eye symptoms (blurred vision caused by mydriasis, diplopia, ptosis, and photophobia), and other cranial nerve signs such as dysarthria, dysphonia, and dysphagia. Symmetrical, descending, progressive flaccid skeletal muscle paralysis follows, with possible abrupt respiratory failure. Laboratory testing is generally not critical to the diagnosis of botulism [18]. The mouse bioassay is the classic test. Competitive reverse transcriptionPCR, which measures the level of toxin-encoding messenger RNA in C. botulinum, is more rapid and sensitive than the bioassay [42]. PCR assays have been used to detect type C1 toxin gene in sediment [43] and type B gene in feces [44] and in food [45]. These molecular techniques are useful for the detection of the infectious forms of botulism in which the toxin is produced by ingested organisms that colonize the digestive tract. In these cases, the organism may be identified in fecal samples. These techniques would be ineffective in classical foodborne botulism, in which the causative agent is preformed toxin and not C. botulinum itself. Com-
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mercial test systems for the identification of anaerobic bacteria may misidentify the C. botulinum strains [46]. Supportive care, including intubation and ventilatory assistance, can reduce the mortality rate significantly. Early administration of antitoxin neutralizes circulating toxin when given during symptom progression. The presence of symptoms indicates circulating toxin, and when symptom progression ceases no circulating toxin remains. Three antitoxins are available in the United States: trivalent equine (types A, B, and E) from the CDC, monovalent human antiserum (type A) from the California Department of Health Services, and a “despeciated” equine heptavalent antitoxin (all seven serotypes) developed by USAMRIID [18]. The heptavalent antitoxin is prepared from horse immunoglobulin G molecules by cleaving the Fc fragments leaving F(ab)2 fragments. No licensed vaccine is currently available. The U.S. army has a pentavalent (types A through E) toxoid, prepared by combining separate aliquots of the 5 inactivated toxins, that is given in a three-dose series (0, 2, and 12 weeks) with annual boosters [47]. This has been given to several thousand volunteers and induces antitoxin levels corresponding to protective levels achieved in experimental animals.
Tularemia F. tularensis, a small, nonmotile, aerobic Gramnegative coccobacillus, causes tularemia. Humans usually acquire tularemia (also known as rabbit fever and deer fly fever) through mucous membrane contact with tissue or fluids of infected animals or bites of infected deer flies, mosquitoes, or ticks. Less frequently, inhalation of contaminated dusts or ingestion of contaminated foods may cause clinical disease. The six forms of tularemia in humans are typhoidal, ulceroglandular, glandular, oculoglandular, oropharyngeal, and pneumonic [18]. Weaponization of the organism would most likely be via aerosol dispersion, with resulting tularemia of the typhoidal and pneumonic type. Onset of symptoms is usually abrupt after a variable incubation period (1 to 21 days, average 1 to 5 days). Manifestations include fever, prostration, weight loss, and pneumonia with nonproductive cough. Tularemia can be diagnosed by identification of the organism from body fluids or by serologic
testing using bacterial agglutination or ELISA. Several PCR assays have been developed for the identification of F. tularensis in body fluids and infected animals and vectors [48–50]. One such assay [50] uses FTA filter paper for specimen preparation and a TaqMan 5' nuclease assay directed against the F. tularensis outer membrane protein (Fop) gene and a PCR–enzyme immunoassay directed against the tul 4 gene. Antibiotic therapy using gentamicin, ciprofloxacin, or streptomycin is effective. An investigational live attenuated vaccine is available for U.S. military personnel [18].
Viral Hemorrhagic Fevers Viral hemorrhagic fever (VHF) syndrome describes the disease processes caused by several RNA viruses from the Filoviridae (Ebola and Marburg), Arenaviridae (Junin, Lassa, Machupo, Guanarito), Bunyaviridae (Nairovirus, Phlebovirus, Hantavirus), and Flaviviridae (Yellow fever virus, Dengue viruses, Kyasanur Forest Disease virus, Omsk hemorrhagic fever virus) families [51]. VHF syndrome is an acute febrile illness characterized by malaise, prostration, generalized signs of vascular permeability, and abnormalities of circulatory regulation. Shock, generalized mucous membrane hemorrhage, and organ involvement often follow; each type has distinguishing clinical features. The viruses are typically transmitted to humans by contact with infected animal reservoirs or arthropod vectors and are highly infectious by aerosol [7]. The filoviruses (Ebola and Marburg) that cause the most tissue destruction and the highest fatality rates among the hemorrhagic fever viruses are discussed here. The genus Filovirus contains four subtypes of Ebola virus (Zaire, Sudan, Ivory Coast, and Reston) and a single species of Marburg virus. Filovirus virions have a distinctive bacilliform shape, but particles can also appear as circular, branched, filamentous, and U- or 6-shaped forms. A dense central core formed by the ribonucleoprotein complex is surrounded by a lipid envelope derived from the host cell plasma membrane. The virion surface has spikes approximately 7 nm in diameter spaced at about 5- to 10-nm intervals [52]. The genomic RNA sequence has been determined for the Ebola Zaire and two strains of Marburg and shown to
Biological Agents as Weapons of Bioterrorism O Broussard
contain approximately 19,000 bp with seven linearly arranged genes [53]. The encoded proteins have been extensively studied, but the complete pathogenic mechanism of the virus is not known. The virion glycoprotein of Ebola Zaire encoded by gene 4 has been identified as the main viral determinant of vascular cell cytotoxicity and injury. Deletion of a serine-threonine–rich, mucinlike domain of the glycoprotein abolished the cytotoxic effects it induced in human endothelial cells [54]. Techniques for the detection of Ebola antigens include ELISA and the more sensitive reverse transcription-PCR assays [55]. An immunohistochemical test using formalin-fixed skin biopsy specimens has been used for the diagnosis of Ebola virus hemorrhagic fever [51]. Advantages of this technique are that the formalin-fixed biopsy samples are not infectious and can be easily collected, handled, and transported. Treatment of infected patients consists primarily of contact isolation and supportive care. Ribavirin, a nonimmunosuppresive nucleoside analog with broad antiviral properties, has been shown to be somewhat effective for treatment of Lassa fever, Argentina hemorrhagic fever, Congo–Crimean hemorrhagic fever, Rift Valley fever, and Bolivian hemorrhagic fever but has poor activity against filoviruses and flaviviruses [7,18]. Available vaccines include yellow fever vaccine and Argentine hemorrhagic fever vaccine, and at least two other vaccines (Rift Valley fever and Hantaan virus vaccines) are currently under investigation [18]. Animals have been successfully immunized against Ebola (rodents) and Marburg (rodents and nonhuman primates) viruses using recombinant vaccines with Venezuelan equine encephalitis virus as a vector [47].
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have diagnostic applications beyond detection of biological weapons. Any attempt to summarize or even present representative examples of current directions here would be futile, but two sources of information for interested readers are the Department of Defense’s Chemical and Biological Defense Program Annual Report to Congress (http: //www.defenselink.mil/pubs/chembio02012000.pdf) and the Web site for the Defense Advanced Research Projects Agency (DARPA; www.darpa.mil). DARPA is the central research and development organization for the Department of Defense, and its Web site contains information on current projects such as summaries and goals of currently funded projects. In the 2000 Report to Congress, the three objectives of the Joint Medical Biological Defense Research Program are to maintain technological capability to meet present requirements and counter future threats; to provide individual-level prevention and protection to preserve fighting strength; and to provide training in medical management of biological casualties to enhance survival and expedite and maximize return to duty. Some of the molecular-related projects mentioned to achieve these objectives include exploitation of molecular science to determine sites, mechanisms of action, and effect of exposure to biological warfare agents; development of multiagent vaccines; and production of a common diagnostic system that can be deployed at forward sites to rapidly analyze clinical samples for the presence of biological warfare agents as well as infectious diseases of military importance. There is no doubt that all of these projects will use molecular techniques and have molecular diagnostic implications. Received April 24, 2001. Received in revised form June 29, 2001. Accepted August 10, 2001.
Future Directions Funding for research and development projects concerning biological weapons has increased tremendously in the past few years. Some of the progress achieved to this point, particularly in the area of early detection of biological agents, has been mentioned in this report, but publication of preliminary data or results is rare. Any information provided on proprietary procedures and instruments is usually deliberately general and incomplete. Products developed as a result of these projects will
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332 Molecular Diagnosis Vol. 6 No. 4 December 2001 4. Okumura T, Takasu N, Ishimatsu S, et al.: Report on 640 victims of the Tokyo subway sarin attack. Ann Emerg Med 1996;28:129–135 5. Committee on Environmental Health and Committee on Infectious Diseases: Chemical–biological terrorism and its impact on children: A subject review. Pediatrics 2000;105:662–670 6. Centers for Disease Control and Prevention: Biological and chemical terrorism: Strategic plan for preparedness and response. Recommendations of the CDC Strategic Planning Workgroup. MMWR Morb Mortal Wkly Rep 2000;49:1–14 7. Franz DR, Jahrling PB, Friedlander AM, et al.: Clinical recognition and management of patients exposed to biological warfare agents. JAMA 1997; 278:399–410 8. Jortani SA, Snyder JW, Valdes R: The role of the clinical laboratory in managing chemical or biological terrorism. Clin Chem 2000;46:1883–1893 9. Kortepeter MG, Parker GW: Potential biological weapons threats. Emerg Infect Dis 1999;5:523–527 10. Cieslak TJ, Eitzen EM: Clinical and epidemiologic principles of anthrax. Emerg Infect Dis 1999;5:552– 555 11. Office of Technology Assessment, US Congress. Proliferation of weapons of mass destruction. Publication OTA-ISC-559. U.S. Government Printing Office, Washington, DC, 1993, pp. 53–55 12. Meselson M, Guilllimin J, Hugh-Jones M, et al.: The Sverdlovsk anthrax outbreak of 1979. Science 1994;266:1202–1208 13. Inglesby TV, Henderson DA, Bartlett JF, et al.: Anthrax as a biological weapon: medical and public health management. JAMA 1999;281:1735–1745 14. Dixon TC, Meselson M, Guillemin J, Hanna PC: Medical progress: Anthrax. N Engl J Med 1999; 341:815–826 15. Pile JC, Malone JD, Eitzen EM, Friedlander AM: Anthrax as a potential biological warfare agent. Arch Intern Med 1998;158:420–434 16. Zoon K: Vaccines, pharmaceutical products, and bioterrorism: Challenges for the U.S. Food and Drug Administration. Emerg Infect Dis 1999;5: 534–536 17. Russell PK: Vaccines in civilian defense against bioterrorism. Emerg Infect Dis 1999;5:531–533 18. Kortepeter M, Christopher G, Cieslak T, et al.: USAMRIID’s medical management of biological casualties handbook, 4th ed. U.S. Government Printing Office, Washington, DC, 2001 (http: //www.usamriid.army.mil/education/bluebook.html) 19. Stepanov AV, Marinin LI, Pomerantsev AP, Staritsin NA: Development of novel vaccines against anthrax in man. J Biotechnol 1996;44:155–160
20. Knisely RF: Selective medium for Bacillus anthracis. J Bacteriol 1996;92:784–786 21. Higgins JA, Ibrahim MS, Knauert FK, et al.: Sensitive and rapid identification of biological threat agents. Ann N Y Acad Sci 1999;894:130–148 22. Ibrahim MS, Lofts RS, Jahrling PB, et al.: Realtime microchip PCR for detecting single-base differences in viral and human DNA. Anal Chem 1998;70:2013–2017 23. Belgrader P, Benett W, Hadley D, et al.: Rapid pathogen detection using a microchip PCR array instrument. Clin Chem 1998;44:2191–2194 24. What’s new in NAI (nonproliferation, arms control, and international security). http://www.llnl. gov/nai/new.html (accessed 4/12/01) 25. Lawrence Livermore National Laboratory: New Technologies Engineering Division. http://www. eng.llnl.gov/documents/eng/EngrME/MENTED/ MRBIG_HANAA.html (accessed 4/12/01) 26. Arita I: Virological evidence for the success of the smallpox eradication programme. Nature 1979;279: 293–298 27. Alibek K: Biohazard. Random House, Inc, New York, NY, 1999 28. Henderson DA, Inglesby TV, Bartlett JG, et al.: Smallpox as a biological weapon: Medical and public health management. JAMA 1999;281:2127– 2137 29. Henderson DA: Smallpox: Clinical and epidemiologic features. Emerg Infect Dis 1999;5:537–539 30. Ropp SL, Knight JC, Massung RF, Esposito JJ: PCR strategy for identification and differentiation of smallpox and other orthopoxviruses. J Clin Microbiol 1995;33:2069–2076 31. Meyer H, Ropp SL, Esposito JJ: Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses. J Virol Methods 1997;64:217–221 32. Neubauer H, Reischl U, Ropp S, Esposito JJ, Wolf H, Meyer H: Specific detection of monkeypox virus by polymerase chain reaction. J Virol Methods 1998;74:201–207 33. Ibrahim MS, Esposito JJ, Jahrling PB, Lofts RS: The potential of 5' nuclease PCR for detecting a single-base polymorphism in orhtopoxvirus. Mol Cell Probes 1997;11:143–147 34. The Johns Hopkins University Center for Civilian Biodefense Studies. http://www.hopkins-biodefense.org/pages/agents/agentsmallpox.html (accessed 4/12/01) 35. The Johns Hopkins University Center for Civilian Biodefense Studies: Meeting of the WHO Variola Research Committee unofficial memo. http://www.hopkins-biodefense.org/pages/news/meeting.html (accessed 4/12/01)
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36. Slack P: The black death past and present. Trans R Soc Trop Med Hyg 1989;83:461–463 37. Inglesby TV, Dennis DT, Henderson DA, et al.: Plague as a biological weapon: medical and public health management. JAMA 2000;283:2281–2290 38. Cornelis GR: Molecular and cell biology aspects of plague. Proc Natl Acad Sci U S A 2000;97:8778– 8783 39. Centers for Disease Control and Prevention: Fatal human plague. MMWR Morb Mortal Wkly Rep 1997;278:380–382 40. Higgins JA, Ezzell J, Hinnebusch BJ, Shipley M, Henchal EA, Ibrahim MS: 5' nuclease PCR assay to detect Yersinia pestis. J Clin Microbiol 1998;36: 2284–2288 41. Cherington M: Clinical spectrum of botulism. Muscle Nerve 1998;21:701–710 42. McGrath S, Dooley JS, Haylock RW: Quantification of Clostridium botulinum toxin gene expression by competitive reverse transcription-PCR. Appl Environ Microbiol 2000;66:1423–1428 43. Williamson JL, Rocke TE, Aiken JM: In situ detection of the Clostridium botulinum type c1 toxin gene in wetland sediments with a nested PCR assay. Appl Environ Microbiol 1999;65:3240–3243 44. Kakinuma H, Muruyama H, Yamakawa K, Nakamura S, Takahashi H: Application of nested polymerase chain reaction for the rapid diagnosis of infant botulism type B. Acta Paediatr Jpn 1997;39: 346–348 45. Aranda E, Rodriguez MM, Asensio MA, Cordoba JJ: Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe. Lett Appl Microbiol 1997;25:186–190 46. Lindstrom MK, Jankol HM, Hielm S, Hyytia EK,
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Biological Agents: Weapons of Warfare and Bioterrorism LARRY A. BROUSSARD, PhD New Orleans, Louisiana
The use of microorganisms as agents of biological warfare is considered inevitable for several reasons, including ease of production and dispersion, delayed onset, ability to cause high rates of morbidity and mortality, and difficulty in diagnosis. Biological agents that have been identified as posing the greatest threat are variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filoviruses (Ebola hemorrrhagic fever and Marburg hemorrhagic fever), and arenaviruses Lassa (Lassa fever) and Junin (Argentine hemorrhagic fever). The pathogenesis, clinical manifestations, diagnosis, and treatment of these agents are discussed. Rapid identification and diagnosis using molecular diagnostic techniques such as PCR is an essential element in the establishment of coordinated laboratory response systems and is the focus of current research and development. Molecular techniques for detection and identification of these organisms are reviewed. Key words: biosensors, anthrax, smallpox, hemorrhagic fever.
increased the fear that the weapons and agents developed may be obtained by, or the personnel involved in these programs may be recruited by, other nations or terrorist groups. The use of Salmonella typhimurium by the Rajneeshee cult in 1984 to intentionally contaminate salad bars in Oregon restaurants [3] and the release of the nerve gas sarin in a Tokyo subway in 1995 by the Aum Shinrikyo cult [4] illustrate the feasibility of use of such agents by groups for political, religious, or other purposes. Recent epidemics of “mad cow” and foot-and-mouth diseases illustrate the potential devastation of antiagriculture biological agents. Depending on the intent of the person, group, or nation using biological agents, the incident may be overt (announced) or covert (unannounced). In either case, if the target is civilian the clinical molecular microbiologist will play a key role in the detection and identification of the agent. For covert incidents, the clinical microbiology laboratory may not only make the initial identification of the organism but also be responsible for recognizing
The terrorist attacks of September 11, 2001 and the subsequent incidents involving anthrax have increased concern about the use of chemical or biological agents by countries, terrorist groups, or individuals. The existence of offensive biological warfare programs in the former Soviet Union and Iraq [1] shows the willingness of some nations to continue such programs even after signing the 1972 Convention on the Prohibition of the Development, Production, and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction (BWC) [2]. The breakup of the Soviet Union and its subsequent economic problems have From the Department of Clinical Laboratory Sciences, Louisiana State University Health Sciences Center, New Orleans, LA. Reprint requests: Larry A. Broussard, PhD, Department of Clinical Laboratory Sciences, Louisiana State University Health Sciences Center, 1900 Gravier St, New Orleans, LA 70112. Email: [email protected] Copyright © 2001 by Churchill Livingstone威 1084-8592/01/0604-0013$35.00/0 doi:10.1054/modi.2001.29155
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the outbreak as an incident of bioterrorism based on the pattern and frequency of symptoms and positive test results. The clinical molecular microbiologist must be familiar with the characteristics of these agents and the technologies available for their detection and identification. If the laboratory does not have the capability to handle and detect these agents, personnel must be familiar with proper handling techniques and have protocols in place for sending the samples to the nearest laboratory with the required capability. The chemical and biological agents that pose the greatest threat to civilian and military populations have been reviewed by several authors [5–9]. Biological warfare agents are easy to manufacture, generally don’t require sophisticated delivery systems, have the ability to kill or injure large populations, cause illnesses that are unrecognized in their initial stages, and may affect large geographical areas if asymptomatic infected individuals travel [5]. Since the termination of the United States offensive biological weapons program by executive orders of President Nixon in 1969 and 1970, many U.S. government agencies have conducted research and developed programs for the detection and identification of biological agents and the prevention and treatment of illnesses caused by these agents. The Centers for Disease Control and Prevention (CDC) Strategic Planning Workgroup recommends that preparedness efforts focus on agents with the potential for the greatest impact on health and security, especially those that are highly contagious or that can be engineered for widespread dissemination via aerosols and have designated these agents as category A [6]. Category A agents include Bacillus anthracis (anthrax), variola major (smallpox), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filoviruses (Ebola and Marburg hemorrhagic fevers), and arenaviruses (Lassa-Lassa fever, Junin-Argentine hemorrhagic fever, and related viruses). The “high priority” biological agents listed in the North Atlantic Treaty Organization (NATO) Handbook on Biological Terrorism include five of the same agents but substitute Brucella species (brucellosis) for the hemorrhagic fever viruses [8]. This article includes a review of the category A agents, including some of the molecular techniques associated with their detection and identification and the development of protective measures against them. These agents include bacteria, a toxin produced by bacteria, a DNA virus, and
RNA viruses. Because similar molecular techniques are often adopted for detection of organisms within the same class (bacteria, RNA virus, DNA virus), each technique is discussed at its first mention. Most of the current research and development is either government funded or performed by the military because of biowarfare implications, but the nonmilitary clinical molecular microbiologist should remain knowledgeable about current developments because of potential terrorist incidents.
Anthrax B. anthracis, an aerobic spore-forming, grampositive rod, causes anthrax (from the Greek for coal because of the black, coal-like cutaneous lesions of the disease). Anthrax spores are very resistant and may remain dormant in the soil for decades. In 1970, a report of the World Health Organization (WHO) estimated that the release of 50 kg of anthrax spores along a 2-km line upwind of a city of 500,000 would result in 95,000 deaths [10], and a 1993 report of the U.S. Congressional Office of Technology Assessment predicted that release of 100 kg of anthrax spores upwind of Washington, DC, would result in 130,000 to 3 million deaths [11]. Germany used B. anthracis to infect livestock and contaminate animal feed to be exported during World War I, Japan used it in World War II, and the organism was included in the programs of the Soviet Union, Iraq, and the United States [2]. The lethal potential of aerosolized anthrax spores was demonstrated by 68 deaths following their accidental release in 1979 from a military facility in Svedlovsk in the former Soviet Union [12]. In humans, anthrax has 3 clinical presentations: cutaneous, gastrointestinal, and inhalational. Cutaneous anthrax, which typically follows exposure to anthrax-infected animals, is the most common naturally occurring form. Gastrointestinal anthrax, which follows ingestion of insufficiently cooked contaminated meats, is fairly uncommon [13]. Inhalational anthrax, known as woolsorters’ disease, was an occupational hazard of workers in slaughterhouses and textile and tanning industries handling contaminated wool, hair, meat, and hides but has been almost completely eliminated in Western nations by immunization. Inhalational anthrax is the form most likely to result from a biological attack. Anthrax spores have a
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diameter of 2 to 6 µm, which is ideal for impinging on human lower respiratory mucosa. After inhalation, spores are phagocytized by tissue macrophages and then germinate into bacilli, which produce a necrotizing hemorrhagic mediastinitis [7]. The time for infection is variable and in Sverdlovsk cases occurred from 2 to 43 days after exposure. Once germination occurs, the disease progresses rapidly. There is no evidence that an immune response is initiated against vegetative bacilli. Anthrax bacilli release two toxins (edema and lethal) leading to hemorrhage, edema, necrosis, and systemic effects that lead to death. Edema toxin consists of protective antigen (PA), which permits entry of the toxin into the host cell, and edema factor, a calmodulin-dependent adenylate cyclase that increases intracellular levels of cyclic adenosine monophosphate, upsetting water homeostasis. Lethal toxin consists of PA and lethal factor, a zinc metalloprotease that inactivates mitogen-activated protein kinase in vitro and stimulates macrophages to release tumor necrosis factor ␣ and interleukin 1 [14]. The exotoxins are thought to inhibit the immune response. The three exotoxin components (PA, lethal factor, and edema factor) are encoded on virulence plasmid pXO1, which is 185.5 kilobase pairs (kbp) in size. The other virulence plasmid, pXO2, is 95.3 kbp and codes for 3 genes (capA, capB, and capC) associated with the synthesis of the polyglutamyl capsule, which inhibits phagocytosis of vegetative anthrax bacilli. Host-specific factors such as temperatures greater than 37°C, CO2 concentration greater than 5%, and the presence of serum components regulate the expression of virulence factors. The transcriptional activator AtxA mediates the regulation of the toxin and capsule genes. The transcriptional activator AcpA also controls expression of the capsule gene. Full virulence requires expression of both plasmids. The Pasteur strain carries pXO2 and does not express exotoxin components; the Sterne strain carries pXO1 but does not have a capsule [14]. The vaccine developed in England is an alumprecipitated cell-free filtrate of a Sterne strain culture and induces measurable antibodies against lethal and edema factors but low levels of antibody to protective antigen [15]. The only licensed vaccine (Bioport Corporation, Lansing, MI) currently available in the United States is an alum-absorbed, partially purified culture filtrate of the avirulent, nonencapsulated strain B. anthracis V770-NP1-R
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[16]. It has been shown to induce high levels of antibody only to PA and requires injections at 0, 2, 4, 6, 12, and 18 months and annually thereafter. Presently all of the available supply is being used for immunization of military personnel. This vaccine is not highly purified and contains multiple extraneous proteins. Current research is focused on developing vaccines with a simpler schedule and better protection. Vaccines now being tested include preparations of PA subunits with different adjuvants, PA purified from recombinant sources, and live vaccines based on anthrax strains with auxotrophic mutations [14]. A vaccine based on purified PA made by recombinant technology has been protective in animals [17]. Inhalational anthrax has initial symptoms (fever, nonproductive cough, malaise, fatigue, and myalgia) similar to those of a viral respiratory infection and is essentially impossible to diagnose in the absence of a known outbreak. Respiratory distress ensues, with shock and death following in less than 24 hours. A widened mediastinum on chest x-ray findings in a previously healthy patient with evidence of overwhelming flulike illness is essentially pathognomonic of advanced inhalational anthrax. Recommended treatment is ciprofloxacin or doxycycline as initial therapy, with penicillin as an alternative once sensitivity data are available [18]. The possibility of genetically engineered resistant strains exists, and there has been one report that Russian scientists developed a strain resistant to tetracycline and penicillin class antibiotics [19]. Laboratory diagnosis of anthrax involves identification of B. anthracis microbiologically, serologically, or by use of more sensitive molecular techniques. B. anthracis forms rough gray-white colonies with characteristic “comet-tail” protrusions on sheep blood agar [15]. It is aerobic, Grampositive, spore-forming, and nonmotile. Polymyxinlysozyme-EDTA-thallous acetate agar is used as a selective medium for B. anthracis if samples are likely to be contaminated with other bacillus species [20]. Although growth should occur within 24 hours, identification of a Bacillus species from a blood culture may require another 24 hours, and the laboratory may not further identify the Bacillus species unless specifically requested to do so [13]. Immunologic tests include specific enzyme-linked immunosorbent assays (ELISAs) to measure antibody titers to PA or to capsular components. Indirect microhemagglutination gives similar results to ELISA but
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has drawbacks, including short shelf life of reagents and longer preparation times [14]. Among the most sensitive methods are the immunomagneticelectrochemiluminescence (ECL) assays for antigen detection and PCR gene amplification for nucleic acid detection [21]. Automated applications are being developed with the ultimate goal of developing portable, hand-held devices capable of rapid detection of all biological threat agents. The ECL system is described in detail by Higgins et al. [21]. Magnetic beads conjugated to a capture antibody and a ruthenium-conjugated detector antibody are used to achieve sensitivities in the range of 0.1 to 1.0 pg/mL. Chemiluminescence is generated by a voltage-dependent reaction of ruthenium heavy metal chelate. This technology is adaptable for use in detection of any biological agent, and its primary limitation is dependence on the availability of high-quality antibodies and other ligands. Detection of protein toxins, not amenable to nucleic acid– based assays, is possible using ECL. Automated ECL detection systems have been developed with the capability of detecting several organisms, including B. anthracis PA antigen. Higgins et al. [21] compared several PCR-based techniques for the detection of pathogenic organisms. It is more difficult to extract DNA from B. anthracis spores than from vegetative cells. Germination of spores for 60 minutes before DNA extraction increased the sensitivity to that achieved with vegetative cells. Among the techniques evaluated for DNA extraction were the Qiagen, Inc (Valencia, CA) kits designed for tissue and blood using protease digestion and chaotropic salt–mediated lysis of cells and the Autolyser (XOHOX Research Institute, Menlo Park, CA) fully automated procedure. The Qiagen system was more sensitive for detection of B. anthracis, but the ease of use of the Autolyser justified further research. Preparation of B. anthracis DNA using FTA paper (Flinders Technologies, Inc, Adelaide, South Australia) and IsoCode cards (Schleicher and Schuell, Keene, NH) was also evaluated. The FTA procedure required several wash steps, compared with the single wash step for the IsoCode paper, and the IsoCode paper was more efficient for plasma or serum matrices. It was determined that the IsoCode extraction could be used to detect B. anthracis in milk at concentrations that may be encountered in a possible bioterrorism scenario [21]. Another technique that has been used for the identification of B. anthracis is the commercial
TaqMan (Perkin-Elmer/Applied Biosystems, Foster City, CA) 5' nuclease assay, which uses fluorogenic probe-based PCR [21]. This technique offers advantages of real-time monitoring, simultaneous assay for different target sequences through use of different oligonucleotide probes labeled with different dye molecules, and the ability to design probes to discriminate between single nucleotide differences in gene sequences. Using this technique, the Special Pathogens Department at the Diagnostic Systems Division of the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) analyzed material from an incident involving a preparation suspected of containing B. anthracis [21]. Assay time was approximately 2 hours. Positive results were obtained when the samples were analyzed with primers and probes for PA gene, and negative results were obtained when the samples were analyzed with primers and probes for capsular antigen gene. The lack of capsular antigen gene, which is associated with virulent strains of B. anthracis, and other data led the investigators to conclude that the sample was an avirulent strain conventionally used in vaccine preparation. Development of portable or handheld instruments for monitoring reactions of fluorogenic probe assays is an ongoing project. The Smartcycler (Cepheid, Sunnyvale, CA) is a commercially available real-time, fluorogenic probe–based PCR portable analyzer. The company plans to develop briefcase-size units [21]. The U.S. Department of Energy’s Lawrence Livermore National Laboratory developed and manufactured a suitcase-size device, MATCI (miniature analytical thermal cycler instrument) that has been used to detect and differentiate between orthopox virus species [22]. The next version of this biodetector was the ANAA (advanced nucleic acid analyzer), which consisted of an array of 10 silicon PCR reaction modules, each containing a silicon PCR microchip, a thermistor to interface the thermal controller with the microchip, and a complete set of optics for fluorogenic detection [23]. This instrument was used to analyze samples of several organisms including Bacillus subtilis spores (to simulate B. anthracis spores) and detection limits of 105 to 107 organisms/L were observed with detection times as short as 16 minutes. The most recent version of the instrument is the HANAA (handheld advanced nucleic acid analyzer), which was introduced in November 1999 and began beta testing by FDA inspectors in July
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2000 [24]. The instrument has four automatically calibrated sample modules and has typical detection times of 6 to 10 minutes [25].
Smallpox Smallpox, a viral disease that once occurred worldwide, exists in two principal forms: variola major and a much milder form, variola minor. Differentiation of the causative viruses is now possible. In 1980, the WHO declared smallpox eradicated after the last natural case of variola occurred in Somalia in 1977 [26]. Following a WHO recommendation that all laboratories destroy their stocks of variola virus or transfer them to one of two WHO-approved repositories, all countries reported compliance. The repositories are the CDC in Atlanta, GA, and the Institute of Virus Preparations in Koltsovo, Novosibirsk region, Russia. An expert committee of the WHO recommended that all viral stocks be destroyed by June 1999, but this action was delayed by another committee’s recommendation in December 1999 that further limited research on variola viruses could be justified but should not extend beyond the end of 2002. The existence of clandestine repositories has been rumored, and a former deputy director of the Soviet Union’s civilian bioweapons program, Ken Alibek, has reported that the Soviet Union has an industrial production capacity of tons of smallpox virus annually and even now has a research program seeking to produce more virulent and contagious recombinant strains [27]. The discontinuation of routine vaccination, high mortality rates (30% or higher), aerosol infectivity, and stability of the virus make it a very dangerous biological weapon threat. Smallpox (variola major), a DNA virus, is a member of the genus orthopoxvirus, which also includes monkeypox, cowpox, and vaccinia. The viruses are complex, and the virion is brick-shaped with a diameter of approximately 200 nm. Spread from person to person occurs via aerosols expelled from the ororpharynx of infected persons, by direct contact, or via contaminated clothing or bed linens. There are no known insect reservoirs or vectors. After aerosol exposure, the virus travels from the respiratory tract to the regional lymph nodes, where it replicates and gives rise to viremia on about the third or fourth day. After multiplication of virus in the spleen, bone marrow, and lymph nodes, a
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secondary viremia begins on approximately the eighth day and is followed by fever and toxemia. After the 12- to 14-day incubation period (range, 7 to 17 days) the patient experiences malaise, fever, rigors, vomiting, headache, and backache [28]. Two or three days later, a papular rash develops over the face and spreads to the extremities. Patients are infectious from the time of onset of the rash and should be isolated until all scabs separate [18]. The rash becomes vesicular, then pustular, and scabs begin to form on approximately the eighth or ninth day of rash. The scabs eventually separate, and pitted scarring develops. For fatal cases, death usually occurs during the second week. All smallpox lesions develop at the same pace, whereas with chickenpox, the disease most commonly confused with smallpox, scabs, vesicles, and pustules may be seen simultaneously on adjacent areas of skin. The chickenpox rash is more dense over the trunk (the reverse of smallpox), and chickenpox lesions are almost never found on the palms or soles [29]. Hemorrhagic and malignant smallpox are two other forms that are difficult to recognize. Hemorrhagic cases are uniformly fatal, and pregnant women appear to be unusually susceptible. The incubation time is somewhat shorter, and the illness is characterized by high fever and head, back, and abdominal pain. A dusky erythema develops, followed by petechiae and frank hemorrhages into the skin and mucous membranes, with death by the fifth or sixth day after onset of rash [28]. The symptoms are similar for the malignant form, which is frequently fatal. In this form, the lesions never progress to the pustular stage and gradually disappear without forming scabs if the patient survives [28]. Laboratory confirmation of a diagnosis of smallpox usually involves identification of the virus from vesicular or pustular fluid collected from scraped lesions using the blunt edge of a scalpel and a cotton swab. State or local health department laboratories should be contacted for shipping instructions, and only high-containment (BL-4) facilities should perform the testing [28]. Preliminary confirmation may include identification of the characteristic brick-shaped virions of orthopoxviruses using electron microscopy. Clinical presentation and medical history help identify cowpox and vaccinia, but differentiation between smallpox and monkeypox virions is virtually impossible without further testing. Ultimate identification and characterization of strains may be accomplished using PCR and
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restriction fragment length polymorphisms (RFLP) [30]. The gene for A-type inclusion body proteins for orthopoxvirus species has been used for the differentiation of these viruses using PCR [31,32]. Monkeypox and vaccinia virus DNAs were detected and differentiated on the basis of a singlebase polymorphism using a fluorogenic 5' nuclease PCR assay [33]. Primers targeting a DNA sequence of the orthopoxvirus hemagglutinin gene and two fluorescently labeled oligonucleotide probes were used in a single-tube assay. Ibrahim et al. [22] demonstrated detection of orthopoxviruses using the battery-powered MATCI. Consensus orthopoxvirus PCR primers were designed to amplify 266 to 281–bp segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus–specific fluorogenic (TaqMan) probe was designed and used to detect a single-base (A/C) substitution within the HA gene. Real-time detection was achieved in less than 1 hour. Currently there is no antiviral medication for use in infected patients. Cidofovir has been shown to have significant in vitro and in vivo activity against Poxviridae [7] and is being considered as a possible therapeutic agent for military personnel. Vaccination within the first 4 days after exposure may prevent or significantly ameliorate illness, and it has been recommended that all exposed persons as well as health care workers and disaster response personnel receive immediate vaccination in the event of an aerosol release of smallpox. A droplet of vaccine is administered in an area approximately 5 mm in diameter by rapid repeated (15 strokes) injection using a bifurcated needle. The site is covered to prevent touching or possibly transferring the virus to other parts of the body [28]. A vesicle typically appears in 5 to 7 days, forms a scab, and gradually heals within 2 weeks. Complications are the lowest of any vaccinia virus strain but may include postvaccinial encephalitis, progressive vaccinia, eczema vaccinatum, and generalized vaccinia [28]. Because of these complications, the Working Group on Civilian Biodefense recommended development of a vaccine from a more attenuated strain that would retain full efficacy [28]. Such a vaccine derived from a Lister strain was developed in Japan in the mid-1970s and given to more than 100,000 persons in Japan. Vaccinia immune globulin is indicated for treatment of complications of the smallpox vaccine. In the United States, approxi-
mately 140,000 vials of vaccine (50 to 60 doses/vial) are stored at the CDC, and it is estimated that 50 to 100 million doses are available worldwide. This vaccine, produced in the 1970s, consists of vaccine virus (New York Board of Health strain) grown on scarified calves [28]. In 2000, the CDC contracted Oravax of Cambridge, MA, to produce 40 million doses of smallpox vaccine with an anticipated delivery of the first production lots in 2004 [34]. The WHO Advisory Committee on Variola Virus Research agreed that determination of the fulllength genome sequences of additional strains might facilitate design of new drugs and better detection probes when it recommended further limited research on variola virus. Complete sequence information is currently available for two Asian variola major strains and alastrim (variola minor), and partial information is available for two African variola major strains, one variola minor strain, and the Butler and Harvey laboratory strains. Strains to be sequenced include Congo 70 (an African variola major strain) and Somalia 77 (a variola minor strain) [35]. The committee believed the use of variola virus for other research was not justified and suggested that use of alternatives such as surrogate orthopoxviruses was sufficient.
Plague Pandemics of plague, the disease caused by the bacteria Y. pestis, have occurred for centuries and caused the death of millions. During the pandemic beginning in 1346, plague, also known as the black death or great pestilence, killed 20 to 30 million Europeans [36]. Plague remains an enzootic infection of rats, ground squirrels, prairie dogs, and other rodents, and human plague occurs most commonly when plague-infected fleas bite humans, who then develop bubonic plague. Outbreaks of plague in China are attributed to plague-infected fleas dropped by a secret branch of the Japanese army, unit 731, during World War II [37]. The U.S. and Soviet biological weapons programs developed techniques to directly aerosolize plague particles that cause pneumonic plague, a highly lethal and potentially contagious form. An attack today would almost certainly occur via aerosol dissemination of Y. pestis, resulting in an outbreak of pneumonic plague.
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Pneumonic plague begins after an incubation period of one to six days, with high fever, chills, headache, and malaise followed by cough with hemoptysis, progressing rapidly to dyspnea, stridor, cyanosis, and death [18]. A 70-kbp virulence plasmid (pYV) enables Y. pestis to survive and multiply in the host. It encodes the Yop virulon, a system consisting of secreted proteins called Yops and their dedicated type III secretion apparatus called Ysc. This Yop virulon allows extracellular Y. pestis to inject the Yops, which block production of proinflammatory cytokines and induce apoptosis of macrophages [38]. Plague should be suspected whenever large numbers of previously healthy individuals develop fulminant Gram-negative pneumonia, especially if hemoptysis is present [18]. Y. pestis shows bipolar (safety pin) staining with Wright, Giemsa, or Wayson stain. It is a lactose nonfermenter, is urease and indole negative, and colonies are initially much smaller than other Enterobacteriaceae. Observable growth on blood or MacConkey agar typically takes 48 hours. Some automated bacterial identification systems may misidentify Y. pestis [39]. Most strains of Y. pestis produce an F1-antigen in vivo, which can be detected in serum samples by immunoassay [18]. A 5' nuclease PCR (TaqMan) assay targeting the plasminogen activator gene (pla) of Y. pestis was developed by Higgins et al. [40]. The assay detected the organism in experimentally infected fleas and monkey blood and oropharyngeal swabs. It has a detection threshold of 2.1 ⫻ 105 copies of the pla target or 1.6 pg of total cell DNA. The portable ANAA microchip PCR array instrument previously described detected Erwinia herbicola vegetative cells, the nonpathogenic simulator of Y. pestis, with a minimum detection limit of 105 cells/L [23]. Pneumonic plague is invariably fatal if antibiotic therapy is delayed more than 1 day after the onset of symptoms. Recommended antibiotic treatment is streptomycin, gentamicin, ciprofloxacin, or doxycycline administered for 10 to 14 days. Doxycycline is recommended for prophylactic treatment of asymptomatic exposed individuals, and alternative antibiotics include ciprofloxacin, tetracycline, and chloramphenicol [18]. No vaccine is currently available for prophylaxis of plague. The United States–licensed formaldehyde-killed whole bacilli vaccine discontinued in 1999 was effective against bubonic plague but not against pneumonic plague [37]. An
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F1–V antigen (fusion protein) vaccine protected mice for a year against an inhalational challenge and is now being tested in primates [18].
Botulism The anaerobic bacteria C. botulinum produce botulinum toxins, proteins of molecular weight approximately 150,000, which are the most toxic compounds known. There are 7 distinct neurotoxins, A through G, produced by different strains of the bacillus. Botulinum toxin is 100,000 times more toxic than sarin. As a biological weapon, botulinum toxins may be aerosolized or used to sabotage food supplies [7]. The botulinum toxins produce a blockade of acetylcholine release. They are zinc-dependent endopeptidases that cleave polypeptides essential for the docking of synaptic vesicles to the presynaptic membrane of the nerve terminal. Neurotoxins A and E target a protein of the presynaptic membrane, SNAP25, and neurotoxin B cleaves VAMP/synaptobrevin, a protein of the neurotransmitter-containing vesicles [41]. Inhalation of the toxins causes the same symptoms as ingestion, although the time course may be different. Onset of symptoms usually occurs 12 to 36 hours after exposure. Initial symptoms include cranial nerve palsies, eye symptoms (blurred vision caused by mydriasis, diplopia, ptosis, and photophobia), and other cranial nerve signs such as dysarthria, dysphonia, and dysphagia. Symmetrical, descending, progressive flaccid skeletal muscle paralysis follows, with possible abrupt respiratory failure. Laboratory testing is generally not critical to the diagnosis of botulism [18]. The mouse bioassay is the classic test. Competitive reverse transcriptionPCR, which measures the level of toxin-encoding messenger RNA in C. botulinum, is more rapid and sensitive than the bioassay [42]. PCR assays have been used to detect type C1 toxin gene in sediment [43] and type B gene in feces [44] and in food [45]. These molecular techniques are useful for the detection of the infectious forms of botulism in which the toxin is produced by ingested organisms that colonize the digestive tract. In these cases, the organism may be identified in fecal samples. These techniques would be ineffective in classical foodborne botulism, in which the causative agent is preformed toxin and not C. botulinum itself. Com-
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mercial test systems for the identification of anaerobic bacteria may misidentify the C. botulinum strains [46]. Supportive care, including intubation and ventilatory assistance, can reduce the mortality rate significantly. Early administration of antitoxin neutralizes circulating toxin when given during symptom progression. The presence of symptoms indicates circulating toxin, and when symptom progression ceases no circulating toxin remains. Three antitoxins are available in the United States: trivalent equine (types A, B, and E) from the CDC, monovalent human antiserum (type A) from the California Department of Health Services, and a “despeciated” equine heptavalent antitoxin (all seven serotypes) developed by USAMRIID [18]. The heptavalent antitoxin is prepared from horse immunoglobulin G molecules by cleaving the Fc fragments leaving F(ab)2 fragments. No licensed vaccine is currently available. The U.S. army has a pentavalent (types A through E) toxoid, prepared by combining separate aliquots of the 5 inactivated toxins, that is given in a three-dose series (0, 2, and 12 weeks) with annual boosters [47]. This has been given to several thousand volunteers and induces antitoxin levels corresponding to protective levels achieved in experimental animals.
Tularemia F. tularensis, a small, nonmotile, aerobic Gramnegative coccobacillus, causes tularemia. Humans usually acquire tularemia (also known as rabbit fever and deer fly fever) through mucous membrane contact with tissue or fluids of infected animals or bites of infected deer flies, mosquitoes, or ticks. Less frequently, inhalation of contaminated dusts or ingestion of contaminated foods may cause clinical disease. The six forms of tularemia in humans are typhoidal, ulceroglandular, glandular, oculoglandular, oropharyngeal, and pneumonic [18]. Weaponization of the organism would most likely be via aerosol dispersion, with resulting tularemia of the typhoidal and pneumonic type. Onset of symptoms is usually abrupt after a variable incubation period (1 to 21 days, average 1 to 5 days). Manifestations include fever, prostration, weight loss, and pneumonia with nonproductive cough. Tularemia can be diagnosed by identification of the organism from body fluids or by serologic
testing using bacterial agglutination or ELISA. Several PCR assays have been developed for the identification of F. tularensis in body fluids and infected animals and vectors [48–50]. One such assay [50] uses FTA filter paper for specimen preparation and a TaqMan 5' nuclease assay directed against the F. tularensis outer membrane protein (Fop) gene and a PCR–enzyme immunoassay directed against the tul 4 gene. Antibiotic therapy using gentamicin, ciprofloxacin, or streptomycin is effective. An investigational live attenuated vaccine is available for U.S. military personnel [18].
Viral Hemorrhagic Fevers Viral hemorrhagic fever (VHF) syndrome describes the disease processes caused by several RNA viruses from the Filoviridae (Ebola and Marburg), Arenaviridae (Junin, Lassa, Machupo, Guanarito), Bunyaviridae (Nairovirus, Phlebovirus, Hantavirus), and Flaviviridae (Yellow fever virus, Dengue viruses, Kyasanur Forest Disease virus, Omsk hemorrhagic fever virus) families [51]. VHF syndrome is an acute febrile illness characterized by malaise, prostration, generalized signs of vascular permeability, and abnormalities of circulatory regulation. Shock, generalized mucous membrane hemorrhage, and organ involvement often follow; each type has distinguishing clinical features. The viruses are typically transmitted to humans by contact with infected animal reservoirs or arthropod vectors and are highly infectious by aerosol [7]. The filoviruses (Ebola and Marburg) that cause the most tissue destruction and the highest fatality rates among the hemorrhagic fever viruses are discussed here. The genus Filovirus contains four subtypes of Ebola virus (Zaire, Sudan, Ivory Coast, and Reston) and a single species of Marburg virus. Filovirus virions have a distinctive bacilliform shape, but particles can also appear as circular, branched, filamentous, and U- or 6-shaped forms. A dense central core formed by the ribonucleoprotein complex is surrounded by a lipid envelope derived from the host cell plasma membrane. The virion surface has spikes approximately 7 nm in diameter spaced at about 5- to 10-nm intervals [52]. The genomic RNA sequence has been determined for the Ebola Zaire and two strains of Marburg and shown to
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contain approximately 19,000 bp with seven linearly arranged genes [53]. The encoded proteins have been extensively studied, but the complete pathogenic mechanism of the virus is not known. The virion glycoprotein of Ebola Zaire encoded by gene 4 has been identified as the main viral determinant of vascular cell cytotoxicity and injury. Deletion of a serine-threonine–rich, mucinlike domain of the glycoprotein abolished the cytotoxic effects it induced in human endothelial cells [54]. Techniques for the detection of Ebola antigens include ELISA and the more sensitive reverse transcription-PCR assays [55]. An immunohistochemical test using formalin-fixed skin biopsy specimens has been used for the diagnosis of Ebola virus hemorrhagic fever [51]. Advantages of this technique are that the formalin-fixed biopsy samples are not infectious and can be easily collected, handled, and transported. Treatment of infected patients consists primarily of contact isolation and supportive care. Ribavirin, a nonimmunosuppresive nucleoside analog with broad antiviral properties, has been shown to be somewhat effective for treatment of Lassa fever, Argentina hemorrhagic fever, Congo–Crimean hemorrhagic fever, Rift Valley fever, and Bolivian hemorrhagic fever but has poor activity against filoviruses and flaviviruses [7,18]. Available vaccines include yellow fever vaccine and Argentine hemorrhagic fever vaccine, and at least two other vaccines (Rift Valley fever and Hantaan virus vaccines) are currently under investigation [18]. Animals have been successfully immunized against Ebola (rodents) and Marburg (rodents and nonhuman primates) viruses using recombinant vaccines with Venezuelan equine encephalitis virus as a vector [47].
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have diagnostic applications beyond detection of biological weapons. Any attempt to summarize or even present representative examples of current directions here would be futile, but two sources of information for interested readers are the Department of Defense’s Chemical and Biological Defense Program Annual Report to Congress (http: //www.defenselink.mil/pubs/chembio02012000.pdf) and the Web site for the Defense Advanced Research Projects Agency (DARPA; www.darpa.mil). DARPA is the central research and development organization for the Department of Defense, and its Web site contains information on current projects such as summaries and goals of currently funded projects. In the 2000 Report to Congress, the three objectives of the Joint Medical Biological Defense Research Program are to maintain technological capability to meet present requirements and counter future threats; to provide individual-level prevention and protection to preserve fighting strength; and to provide training in medical management of biological casualties to enhance survival and expedite and maximize return to duty. Some of the molecular-related projects mentioned to achieve these objectives include exploitation of molecular science to determine sites, mechanisms of action, and effect of exposure to biological warfare agents; development of multiagent vaccines; and production of a common diagnostic system that can be deployed at forward sites to rapidly analyze clinical samples for the presence of biological warfare agents as well as infectious diseases of military importance. There is no doubt that all of these projects will use molecular techniques and have molecular diagnostic implications. Received April 24, 2001. Received in revised form June 29, 2001. Accepted August 10, 2001.
Future Directions Funding for research and development projects concerning biological weapons has increased tremendously in the past few years. Some of the progress achieved to this point, particularly in the area of early detection of biological agents, has been mentioned in this report, but publication of preliminary data or results is rare. Any information provided on proprietary procedures and instruments is usually deliberately general and incomplete. Products developed as a result of these projects will
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