Ebola virus as a biological warfare agent

Botulinum toxin. The most toxic substance known is botulinum toxin. It is made by the anaerobic bacterium, Clostridium botulinum, and is the cause of botulism, a severe form of food poisoning. It has been proposed as a biological warfare agent but has actually found its most frequent application in cosmetics, under the name Botox.

It is also used to treat a few clinical conditions in which a muscle relaxant is needed. Botulinum toxin is a neurotoxin that blocks transmission of signals from nerves to muscles, thus causing muscular paralysis. The incredible potency of botulinum toxin is due to its enzymatic activity. It is a zinc protease that cleaves SNARE proteins in the neuromuscular junction that are required for release of the neurotransmitter acetylcholine.

Death is generally due to paralysis of the lungs and respiratory failure Fig. Botulinum toxin disrupts the normal functioning of a neuromuscular junction by inhibiting the release of acetylcholine.

Proper canning uses a pressure cooker to destroy the hardy spores produced by Clostridium. If the spores are not destroyed, they can germinate. After the bacteria die, they release botulinum toxin, which accumulates in the food. Merely 50 ng of botulinum toxin is enough to kill the average human. The toxin can, however, be destroyed by heating.

Terrorists of the Japanese cult Aum Shinrikyo, discussed previously, have attempted to use botulinum toxin. Aerosols were dispersed at various sites in Tokyo and at U.

The attacks failed, mainly because the cult used strains of C. On the other hand, millions of people have willingly had extremely dilute preparations of botulinum toxin Botox injected into their face to eliminate wrinkles.

The procedure works because botulinum toxin inhibits the muscle contraction responsible for causing them. Many higher plants make ribosome-inactivating proteins RIPs. These enzymes split the N-glycosidic bond between adenine and ribose from a specific sequence in the large-subunit ribosomal RNA.

Clipping adenine from the rRNA totally inactivates the ribosome. A single RIP molecule is sufficient to inactivate all the ribosomes and kill a whole cell. Intact ribosomes from different types of organisms differ greatly in their sensitivity to RIPs. Mammalian ribosomes which contain 28S rRNA are by far the most sensitive.

Like many bacterial toxins, ricin is a typical A-B toxin in which the A chain exhibits toxic enzymatic activity and the B chain mediates entry into the target cell. Ricin is extracted from the seeds of the castor bean plant, Ricinus communis Fig. This plant is widely grown, both for ornamentation and on a large scale for castor oil production. Because of its widespread availability, high toxicity, stability, and lack of any antidote, there are several examples of the use of ricin as a biological weapon.

Ricin achieved international notoriety in when the Bulgarian defector Georgi Markov was assassinated in a London street by ricin. The communist assassin wielded a modified umbrella that injected a hollow 0.

In , four members of the Patriots Council, an extremist group in Minnesota with an antigovernment and antitax ideology, purified ricin in a home laboratory. They were arrested for plotting to kill IRS and law enforcement agents with ricin. In late , actress Shannon Richardson pleaded guilty for mailing letters containing ricin to President Barack Obama and New York City Mayor Michael Bloomberg in a scheme to frame her estranged husband.

In a completely separate incident, in January , James Dutschke also pleaded guilty to sending ricin to President Obama and other government officials with the intention of framing an Elvis Presley impersonator with whom he had a personal feud.

Though less well known, abrin , which is also a ribosome-inactivating protein, is four times more toxic than ricin. Abrin is derived from the seeds of Abrus precatorius , commonly known as jequirity or rosary pea Fig. The beautiful seeds are widely used in jewelry, particularly rosary beads. However, the seeds are so toxic that, if broken or damaged, a small prick in the skin is sufficient to absorb a lethal dose of abrin.

There have been reports of abrin poisoning in jewelry makers, as well as in individuals who ingested seeds, but there are no known instances of abrin being used as a biological weapon. Cone snails are predators that use a venom cocktail containing at least different conotoxins to paralyze and kill their prey. Other toxins may trigger cardiovascular collapse. Because most conotoxins are short peptides 10—30 amino acids in length, the concern from a biological warfare perspective is not that a government or terrorist would harvest venom from cone snails but that the toxins would be chemically synthesized.

Purified toxins are possible biological warfare agents. Natural toxins can be isolated from bacteria, plants, and animals, but other toxins could be chemically synthesized. Botulinum toxin disrupts the neuromuscular junction and is the most potent toxin known. Ricin and abrin are ribosome-inactivating proteins that are made by certain plants. It is often suggested that genetic engineering could be used to create more dangerous versions of infectious agents. Although there is some truth to this assertion, consider the following:.

Suppose a bioterrorist tries to genetically modify a harmless laboratory bacterium, such as E. Additionally, the bacteria could be programmed to rebuff immune cells by injecting them with toxins, and other genes could be added for ripping vital supplies of iron away from blood cells. Finally, the bacteria could be modified to be highly infectious. Such a biological agent would make for a fearsome weapon. Unfortunately, this bacterium already exists.

It is called Yersinia pestis. It is the agent of bubonic plague and is still endemic in many parts of the world, including China, India, Madagascar, and the United States. Still, genetic engineering of biological warfare agents is theoretically possible, so we briefly consider the issue here. The Soviet germ warfare facility is known to have modified smallpox virus and generated a variety of artificial mutants and hybrids.

The details are largely unavailable. However, recent experiments with mousepox Ectromelia virus have given disturbing results. Mousepox is related to smallpox, but it only infects mice. Its virulence varies greatly depending on the strain of mouse. Genetically resistant mice rely on cell-mediated immunity, rather than antibodies. Natural killer NK cells and cytotoxic T cells destroy cells infected with mousepox virus, thus clearing the virus from the body.

Researchers modified mousepox virus by inserting the human gene for the cytokine interleukin-4 IL IL-4 is known to stimulate the division of B cells, which synthesize antibodies. The rationale for engineering the virus was that IL-4 would stimulate the production of antibodies and lead to an improved and more balanced immune response. What actually happened was the opposite of what was expected: the creation of a virus with vastly greater virulence. Furthermore, it failed to increase the antibody response.

The reasons are not fully understood, but they do serve as a reminder that the immune system is under extremely complex control. Similar results have been seen with strains of Vaccinia virus, which is used for vaccination against smallpox.

Whether insertion of IL-4 or other immune regulators into smallpox itself would lead to increased virulence by undermining the immune response is unknown. These are the cytokine response modifier crm genes, and they vary in effectiveness among different poxviruses.

In this case, adding IL-4 would not be expected to increase virulence. With genetic engineering, it is also possible to hide a potentially dangerous virus inside a harmless bacterium.

This strategy is already used in nature when bacteriophages insert their genomes into bacterial chromosomes or plasmids and later re-emerge to infect other hosts. Theoretically, cloning the entire genome of a small animal or plant virus into a bacterial plasmid could create a biological weapon.

Larger viruses could be accommodated with bacterial or yeast artificial chromosomes. In the case of RNA viruses, a cDNA copy of the virus genome must first be generated by reverse transcriptase before cloning it into a bacterial vector.

Any virus containing a poison sequence , a base sequence that is not stably maintained on bacterial plasmids, could perhaps be cloned as separate fragments.

Such a strategy works for yellow fever virus, but a complete, functional cDNA requires ligation of the fragments in vitro. Consequently, the naked nucleic acid genomes of many viruses, both DNA and RNA, are infectious even in the absence of their protein capsids or envelopes. Thus, once a viral genome is cloned, the DNA molecule containing it may itself be infectious.

This has been demonstrated for RNA viruses such as poliovirus, influenza, and coronavirus. The cleverest strategy for generating an RNA virus is to clone the cDNA version of its genome onto a bacterial plasmid downstream of a strong promoter Fig. The natural RNA version of the viral genome will be generated by transcription.

When induced, the bacterial cell would generate a large number of infectious viral particles. A dangerous human RNA virus loaded into a harmless intestinal bacterium under the control of a promoter designed to respond to conditions inside the intestine could pose a formidable threat. Genetically engineering biological warfare agents to make them deadlier is a minor threat since many naturally occurring microbes are already very dangerous. However, certain poxviruses have been modified to become more virulent.

Inserting viral DNA into plasmids carried by harmless bacteria could create camouflaged viruses. The cDNA is inserted into an appropriate bacterial plasmid and transformed into bacterial cells.

If the promoter is inducible, when the bacteria are given the appropriate stimulus, the viral cDNA will be expressed, resulting in production of viral particles that could infect many people. In the laboratory, some pathogenic bacteria grow slowly or not at all. This may be because the microbe has fastidious nutrient requirements or is otherwise difficult to culture outside its host organism. However, thanks to advances in biotechnology, infectious microbes can be identified using a variety of different techniques.

Rather than attempting to grow and identify disease-causing agents using classical microbiological techniques, molecular diagnostics analyzes molecules; typically DNA, but RNA, proteins, and volatile organic compounds can also be used. Other diagnostic methods involve the use of antibody technology and are discussed in Chapter 6. Molecular techniques have the advantage of being quicker, more accurate, and more sensitive.

Biopsies or other patient samples are directly probed with fluorescent DNA oligonucleotides specific to a pathogen of interest. If the pathogen is present, the probe binds to the complementary DNA in its chromosome and the fluorescence can be visualized under a microscope.

A new innovation, called peptide nucleic acid PNA , replaces the negatively charged sugar-phosphate backbone of DNA with a neutral peptide backbone.

Fluorescence microscopy; original magnification x. Because primers can be designed to amplify DNA sequences unique to a particular pathogen, PCR itself can serve as a diagnostic tool. The advantages of PCR are that it theoretically requires only a single molecule of target DNA and works on microbes that cannot be cultured in the laboratory. The downside is that PCR is susceptible to contamination and false positives.

This capability is useful in epidemiology for tracking the spread of infectious disease. This allows multiple bacteria to be detected and identified simultaneously in a single sample.

A series of probes corresponding to different bacteria are applied in horizontal lines across a hybridization membrane Fig. The PCR fragments are then labeled with a fluorescent dye and applied vertically to the membrane. After denaturation and annealing to allow hybridization, the membrane is washed to remove unbound DNA. Those samples that hybridize to the probes appear as bright fluorescent spots. Probes corresponding to 16S rRNA for each candidate bacterium are attached to a membrane filter in long horizontal stripes one candidate per stripe.

The PCR fragments are tagged with a fluorescent dye and applied in vertical stripes. Each sample is thus exposed to each probe. Wherever a 16S PCR fragment matches a 16S probe, the two bind, forming a strong fluorescent signal where the two stripes intersect. It combines traditional PCR with mass spectrometry to identify unknown microbes in patient samples. DNA is extracted and many different sets of primers are used to amplify various target sequences.

The fragments are then analyzed with a mass spectrometer to determine their mass. From this information, the DNA sequence can be deduced and the pathogen identified. Biosensors are devices for the detection and measurement of reactions that rely on a biological mechanism Fig. Biosensors have been traditionally used in medical diagnostics and in food and environmental analysis. By far the biggest use has been the clinical monitoring of glucose levels in diabetics using the enzyme glucose oxidase.

Biosensors, in general, share a common design. A highly specific biological receptor molecule detects or interacts with a target molecule of interest, for instance, a biological warfare agent.

A signal is generated, processed, and displayed for the user. There is growing interest today in using biosensors to detect biological warfare agents. Placing biosensors in high trafficked areas, such as in malls or subway stations, could allow for continuous surveillance. Additionally, handheld devices giving a rapid response at the site of a possible attack would be highly useful. Several proposals exist that would use specific antibodies or antibody fragments as detectors for biological warfare agents see Chapter 6 for antibody engineering.

B cells carry antibodies specific for one antigen, so one proposal is to use whole B cells in a biosensor. When an antigen binds to the antibody on the surface of a B cell, it triggers a signal cascade. Engineered B cells have been made that express aequorin , a light-emitting protein from the luminescent jellyfish Aequorea victoria. Aequorin emits blue light when triggered by calcium ions Fig. Living jellyfish actually produce flashes of blue light, which are transduced to green by the famous green fluorescent protein GFP.

Aequorin, from Aequorea victoria, emits blue light when provided with its substrate, coelenterazine, plus oxygen and calcium. The enzyme binds to aequorin via the oxygen; and when calcium is present, the complex emits blue light, degrades the substrate to coelenteramide, and releases carbon dioxide. In a biosensor, when a B cell detected a disease agent or any specific antigen , calcium ions would flood into the cell due to activation of a signal cascade Fig. This in turn triggers light emission by aequorin.

The light emitted is detected by a sensitive charge-coupled device CCD detector. This approach could detect 5 to 10 particles of a biological warfare agent. Approximately 10, B cells specific to different pathogens could be assembled in array fashion onto a chip placed inside the biosensor. Expressing aequorin in a B cell would provide a detection system for B-cell activation.

When a trigger molecule, such as a biological warfare agent, binds to receptors on the B cell, the calcium channels are opened and calcium floods the cell.

The high calcium levels would activate aequorin to emit blue light. A charge-coupled device CCD would measure the photon emissions and warn the user of a biological agent. Another scheme developed by the Ambri Corporation of Australia uses antibody fragments mounted on an artificial biological membrane, which is attached to a solid support covered by a gold electrode layer. Channels for sodium ions are incorporated into the membrane.

When the ion channels are open, sodium ions flow across the membrane and a current is generated in the gold electrode. The ion channels consist of two modules, each spanning half the membrane. When top and bottom modules are united, the ion channel is open. When the top module is pulled away, the ion channel cannot operate. Binding of biological warfare agents by the antibody fragments separates the two halves of the channels, which in turn affects the electrical signal Fig.

Diagnosing pathogenic bacteria with molecular techniques, particularly using the genes encoding ribosomal RNA sequences, is faster and more sensitive than traditional microbiological methods. Biosensors use biological components themselves to monitor for suspicious biomolecules. Antibody fragments that bind specific biological agents can be engineered and tethered to a fixed location on an artificial membrane. Another molecule of the same antibody fragment is tethered to a sodium channel.

The artificial membrane is carried on a gold-coated solid support that acts as an electrode. This detects sodium ions that pass through the ion channel. When a biological agent is present, the antibody fragments bind it, pulling the top half of the sodium channel out of alignment with the bottom half.

Sodium ions no longer pass to the gold electrode, decreasing the signal. Biological warfare has been around since life first evolved. Humans are most concerned about the biological warfare directed at us, including infectious diseases and the ability of microbes to evolve resistance to antibacterial agents. Although this development is worrisome, new strategies and technologies are being developed to fight back against the growing problem of antibiotic resistance.

Humans have often attempted to use biological agents in warfare, although with little overall success so far. Several highly virulent infectious agents including anthrax, plague, and smallpox, as well as certain biological toxins such as ricin and abrin, are regarded as likely biological warfare agents.

Developing quicker ways to detect and diagnose microbes is an active area of research. Siderophores are a good target because they do not exist in humans, and thus the side effects would be diminished. Once the virus had been identified and patients isolated, the pathogen would have been unlikely to spread widely.

Still, any terrorist attempting to stoke fears rather than accrue a high body count could have some modicum of success with Ebola. Interviews with Fauci and other infection and security experts suggest that the virus could potentially be used for small-scale Ebola attacks in about three different ways—although each approach would run up against substantial logistical, financial and biological barriers.

Ebola would not need to be altered in any way to make such a plot work. The virus is already so capable of spreading from person to person via contact with bodily fluids that in its natural state it could do some serious damage. Such a plan would hinge on injecting Ebola virus into a limited number of people, who would then need to leave west Africa or wherever the outbreak may be before becoming symptomatic.

Then those individuals would have to get into a public space and projectile vomit or bleed onto others to infect them. Obviously the plot would need to overcome substantial technical challenges including the extreme weakness that arises from Ebola.

If it did succeed, this mode of transmission would not kill thousands of people, but it would set off significant fears. The third bioterrorism method appears to be the most unlikely: genetically modifying the virus to enable it to spread more readily, perhaps through the air. Classified by the CDC as a category A biological agent, Ebola virus causes severe hemorrhagic fever, characterized by high case-fatality rate; to date, no vaccine or approved therapy is available.

The EVD epidemic, which broke out in West Africa since the late , has got the issue of the possible use of Ebola virus as biological warfare agent BWA to come to the fore once again. It began on September 1st with a year-old male Mission school teacher who sought treatment for what he thought was a case of malaria at the Yambuku Mission Hospital.

He received an injection of malaria medication, and the Ebola virus spread through medical equipment that was no sterilized. The last case died on November 5th. One case was discovered in Tandala, Zaire, in The next major outbreak of Ebola-Zaire was in Gabon in in the Mekouka and other gold-mining camps in the deep rain forests.

In , a severe outbreak of Ebola-Zaire began in Kikwit, Zaire, beginning with a charcoal worker on January 6th. The disease spread by person-to-person contact and through ritual cleansings of the victims' bodies before burial.

The outbreak officially ended on August 24th. In , an outbreak in the Mayibout area of Gabon occurred following the ingestion of a dead chimpanzee found in the forest. A similar outbreak occurred in in the Booue area of Gabon that spread from a hunter who lived in a forest camp.