Changing the Recipe-Genetic Engineering and Biological Weapons

     Genetic engineering or genetic modification is a technique which involves the direct manipulation of an organism's genome using modern biotechnology. The process changes the genetic makeup of an organism, removing or adding to the organism's genetic material (DNA).

Genetic Engineering

     Artificial selection (also known as selective breeding) involves intentional breeding of animals or plants for certain traits, or combination of traits and has been carried out for thousands of years. This is different from Darwin's natural selection where the environment selects out or adds individual traits which may be needed for better survival. But artificial selection can also be unintentional. For example, it is believed that domestication of crops (such as certain grains) by early humans was largely unintentional, occurring by chance as the best seeds tended to survive or provide better nutrition.

     Mutagenesis is another process that can be used to change the genetic information of an organism. Alteration of a gene or group of genes may occur spontaneously in nature or can be a result of exposure to mutagens, chemicals or radiation which induce the changes in the nuclei of the cell where the DNA is held.

The Development of Maize-
Artificial Selection?

     It is a technique which can also be achieved using laboratory procedures, creating genetic changes in the organism. In nature, mutagenesis can lead to cancer and various heritable disease but it is also the driving force of evolution.

     Genetic engineering, mutagenesis and artificial selection are the three techniques used by biologists seeking to create the ultimate biological weapon, a weapon which has no cure, no vaccine to protect against it or even a weapon which can selectively attack only certain members of the human species.

     But the creation of a biological weapon does not require genetic engineering. There are many deadly 'natural' organisms which are readily available to those who are determined to obtain them (smallpox, anthrax). However, the expansion of modern biotechnology in the medical and pharmacological fields as well as means of production has led to an easy availability of knowledge and facilities.

     Many countries which had previously been able to access only rudimentary technology, can now access high-tech facilities for vaccine or organism production that could be easily be altered for the production of biological weapons.

     Today, nearly all countries have the technological potential to produce large amounts of pathogenic microorganisms safely. 'Classical' biological agents can often be made more efficiently today than their natural counterparts using even simple genetic techniques. With this modern biotechnology, it is possible to create completely new biological weapons.

     Not only can artificial organisms be created which are resistant to antibiotics and vaccines but these new organisms are also more toxic, harder to detect and more stable in the environment.

     The use of genetic engineering in the development of biological weapons is not merely a theoretical possibility. Genetic engineering has been used in the former Soviet Union, with the creation of the USSR's 'invisible anthrax' where Soviet scientists inserted an 'alien' gene into Bacillus anthracis, altering its immunological properties. Vaccines against this new strain of anthrax proved to be ineffective.

     Anthrax is a prime choice as a biological weapon since it ban be produced in large amounts, acts rapidly and remains robust in the environment, surviving years buried in the soil (see post: Biological Weapons - How Bad Can They Be?). Ideally, the disease must be treatable, or a vaccine must be available for one's own troops.  However, potential victims of an anthrax attack can be treated with antibiotics even several days after an infection and, in most circumstances, only a minority of the infected die from anthrax. But just a very simple genetic intervention, such as that carried out by the Soviet Union, is often enough to 'change the rules of the game'  producing (as in the Soviet case) a form , resistant to vaccine and possibly even a variant resistant to antibiotics.

     There are a number of different approaches to the development of a 'synthetic' biological weapon, that is a natural disease which has been genetically altered or'enhanced'.

State Research Center for
Applied Microbiology, Obolensk

     Development of symptom-altered biological agents is one method of choice. Researchers from the State Research Center for Applied Microbiology in Obolensk, (about 85 kilometers south of Moscow) inserted a gene into Francisella tularensis (the bactrium which causes tularemia), making the bacteria produce beta-endorphin, an endogenous human drug (see post: Your Personal Narcotic). This genetic alteration caused changes in the behaviour of mice infected with this transgenic bacteria.

     Supposedly, the endorphin gene was not introduced into a fully virulent strain, but only into a vaccine strain. If inserted into virulent F. tularensis, the victims would not have shown the usual symptoms of tularemia, but instead unusual symptoms which would, in theory at least, have obscured the diagnosis and delayed therapy.

Tularemia

     The transfer of a lethal factor to harmless human gut bacteria through genetic engineering could also transform previously harmless bacteria into lethal biological weapons. This was actually achieved by US researchers in 1986. They isolated the gene for the lethal factor of Bacillus anthracis (the causative agent of anthrax) and introduced into Escherichia coli (a normal and harmless resident of the human gut).
     It was reported that the lethal factor protein was active in E. coli and displayed the same deadly effects as it did when in its native B. anthracis.

     There have been a number of countries which have attempted to create antibiotic resistant anthrax and tularemia.

     German researchers at the Santitaetsakademie der Bundeswehr in Munich cultured genetically engineered Francisella tularensis subsp. holarctica bacteria, a close relative of the causative agent of tularaemia. An antibiotic resistance marker gene (tetracyclin) was been inserted into these bacteria.

Porton Down

     British researchers from Porton Down in the UK used genes conferring resistance to antibiotics for genetic studies in fully virulent strains of anthrax.
     In the 1980s, a researcher at the University of Massaschussetts introduced antibiotic resistance genes into anthrax, rendering it less treatable with antibiotics.

     Researchers from the Institut Pasteur in Paris did similar work as their counterparts in Russia, introducing antibiotic resistance genes into anthrax bacteria.

     Detection of biological weapons depends on molecular recognition of the microbe using antibodies similar to the human immune system. Altering the immunogenicity of the microorganism not only overcomes vaccinations but also the detection systems.

Institut Pasteur

     In 1997, the same Russian research group from Obolensk published a paper on another effort to genetically engineer anthrax.  The team was able to put new genes into fully pathogenic strains of anthrax and alter the bacteria's immunopathogenic properties, making existing anthrax vaccines ineffective against the new genetically-engineered types.

     The Russian researchers also constructed a new vaccine against the new strain which potentially enabled the army using such a weapon, vaccination of their own soldiers against a specific strain, while the enemy remained vulnerable.

State University of New York in Stony Brook

      In 2002, a research team at the State University of New York in Stony Brook chemically synthesized an artificial polio virus from scratch  beginning with the genetic sequence of the agent which was available online. They ordered small, tailor-made DNA sequences and combined them to reconstruct the complete viral genome. Then, the synthesized DNA was brought to life by adding a chemical cocktail that initiated the production of a living, pathogenic virus.

     Many countries (such as the US) have investigated the development of material-degrading microorganisms aimed at destroying fuel, construction material or stealth paints. Many natural microorganisms are able to degrade nearly every material and are already being used to detoxify environmental pollution. The natural organisms, however, are slow-acting and unreliable, but, with the help of genetic engineering, the development of much more effective organisms might become possible—probably effective enough to be used as biological weapons.

     These investigations have been sparked by renewed interest in non-lethal weapons for use in media-sensitive military operations so that visible civilian victims can be avoided.

     In 1998, the US Naval Research Laboratory in Washington DC was declared to be developing genetically engineered fungi with offensive biological warfare potential. Researchers were able to isolate natural microorganisms which degrade a variety of materials, such as plastics, rubber and metals and used genetic engineering to make them more powerful and focused. One of these genetically engineered microbes apparently can destroy military paints in 72 hours.
     James Campbell, principal investigator at the Naval Research Laboratory, noted possible applications of this technology including microbial derived or based esterases, capable of stripping signature-control coatings from aircraft, allowing easier identification of enemy rockets or airplanes.

US Naval Research Laboratory

     The USA also increased efforts to identify microorganisms that kill drug-producing crops and, by the late 1990s, this research was focused on two fungi. The organism, Pleospora papaveracea, a killer of the opium poppy, was conducted in Tashkent, Uzbekistan, with financial and scientific support from the USA.

     Pathogenic Fusarium oxysporum strains developed in the USA to kill coca plants were scheduled for field tests in Colombia in 2000. These two species of fungi provide an excellent example of the hostile use of biological agents. In Colombia, the biggest areas of coca and opium poppy cultivation are in combat zones, and the 'War on Drugs' is part of that country's continuing armed conflict.

Pleospora papaveracea

     Some claim that the pursuit of crop-killing fungi as weapons would be a further slide down a slippery slope that, by following the same logic, could easily lead to the use of other plant pathogens, animal pathogens or even non-lethal biological weapons against humans.

     Another example of new ideas in warfare concern substances which are not necessarily biological weapons but new types of chemical, or rather biochemical weapons.  This area came under the spotlight of the international media after the use of psychoactive substances in the Moscow hostage crisis (Nord-Ost Siege) in October 2002, causing the death of more than 170 people.

     The chemicals used by the Russian 'swat' team to free the hostages held by Chechen rebels were probably narcotic based but the authorities refused to divulge the exact nature of the chemicals used.

     Many of these supposedly 'non-lethal' chemical weapons had been developed as early as the 1950s, particularly a substance called 'BZ (3-quinuclidinyl benzilate)', known in the US army as 'sleeping gas' and more commonly as 'buzz' because of its abbreviation as well its effects on the mental state of its victims.

Nord-Ost Siege (October 2002)

     BZ, invented by Hoffman-Laroche (see post: The Dawn of the Drug Dealers) in 1951,  is an anticholinergic compound (similar to a neurotransmitter-see post: The Genetics of Drug Addiction) related to scopolamine, atropine and other similar chemicals. Dispersal could be carried out as an aerosolized solid for breathing in or as agent dissolved in one or more solvents for ingestion or absorption through the skin.

     In 1998, the British accused Iraq of having stockpiled large amounts of a substance called Agent 15, a glycolate anticholinergic incapacitating agent chemically either identical to BZ or closely related to it. Agent 15 was reportedly stockpiled in large quantities prior to and during the First Gulf War.

Project SHAD

     During the Cold War in the US, Project SHAD (Shipboard Hazard and Defense)  a project under the guise of the Department of Defense was a series of tests carried out to investigate the use of both chemical and biological weapons related to a program dubbed, Project 112. Also known as the Edgewood Arsenal experiments, Project 112 investigations were said to part of CIA mind-control programs started after WWII.
     The experiments were performed at the Edgewood Arsenal, northeast of Baltimore, Maryland, and involved the use of hallucinogens such THC, LSD, BZ and other chemical and biological agents.

     BZ is odorless, very stable in most solvents and has a half-life of 3-4 weeks in moist air. It is persistent in soil, water and on most surfaces. BZ can be synthesized in clandestine laboratories but its recreational use is almost nonexistent, because of its unpleasant effects.
     Because BZ is odorless and nonirritating with delayed symptoms for several hours after contact, the only immediate indications of its use may be the white smoke emanating from delivered weapons.

     There is little risk of absorption f BZ through the skin or contact hazards from aerosols that have settled out onto exposed surfaces. The most protective response is to don a protective mask with a good quality aerosol filter. There is the possibility, however, that BZ could be employed to render activity through skin by the addition of a skin penetrating solvent or by using a secondary aerosol, contaminating the surrounding terrain with BZ in bulk micro-pulverized form.

BZ (3-quinuclidinyl benzilate)

     BZ is distributed to most organs and biological tissues of the body and is able to cross the blood-brain barrier, conferring effects upon the central nervous system.

     BZ an incapacitating rather than a toxic warfare agent and acts as a competitive inhibitor (blocker) of the neurotransmitter acetylcholine neurons.  As the concentration of BZ at these sites increases, the proportion of receptors available for binding to real acetylcholine decreases and the end organ 'sees' less acetylcholine. This leads to clinical effects reflective of under-stimulation of end organs, such as blurry vision, dry mouth and skin, decreased sweating and dilated pupils. With decreased sweating, the body core temperature rises. Effects on smooth muscle include decreased bladder tone with possibly severe bladder distension.

     BZ typically raises the heart rate initially, but hours later, depending on the dose of BZ, the heart rate falls to normal or may become slow.

     Central nervous system effects are more profound and include a decrease in the level of consciousness, beginning with drowsiness and progressing through sedation to coma. The patient is often disoriented to time and place with disturbed abilities in judgment and insight. The patient may abandon socially imposed restraints and resort to vulgar and inappropriate behavior. Perceptual clues may no longer be readily interpretable, and the patient is easily distracted and may have memory loss, especially short-term memory.

     The victim may also experience including illusions (misidentification of real objects) and hallucinations (the perception of objects or attributes that have no objective reality). Another effect of BZ poisoning is behavioral lability, with patients swinging back and forth between quiet confusion and self-absorption in hallucinations, to frank combativeness. Moreover, as other symptoms begin to resolve, intermittent paranoia may appear. Automatic behaviors common during resolution include crawling or climbing motions, previously called 'progresso obstinato' in old texts describing dementia.

Mass Hysteria

     BZ can also produce 'shared' illusions and hallucinations (folie en famille, folie a deux, mass hysteria). An examples of this is a case where two individuals took turns smoking an imaginary cigarette clearly visible to both of them but to no one else and another case where two victims of BZ played tennis with imaginary rockets.

     The course from BZ poisoning takes place in four stages. Stage 1 begins within 30 minutes to 4 hours after exposure and is characterized by parasympathetic blockade (dryness, blurry vision, etc.).
     The second stage (4 to 20 hours after exposure) is characterized by stupor with loss of balance and increased body temperature.
     Stage 3 (20 to 96 hours after exposure) produces full-blown delirium.
     The fourth stage or resolution stage, is characterized by paranoia, deep sleep, reawakening, crawling or climbing automatisms, and eventual reorientation.

     But because BZ can cause very different effects in different individuals, it was considered to be unreliable, leading to the banishment of this substance from the US chemical arsenal in the late 1960s.

     More efficient classical biological warfare agents will probably have only a marginal role, even if the genetically engineered 'superbug' is still routinely featured in newspaper reports. More likely and more alarming are weapons for new types of conflicts and 'niche' warfare scenarios, such as or covert operations,  economic warfare or sabotage activities.

     Biological organisms can be modified in a variety of different ways. Toxins, for instance, can be produced by adding the DNA coding for its production to previously harmless bacteria. Advances in biotechnology have made it possible to synthesize certain viruses based on its genome (the organism's genetic instructions) and using basic materials such as DNA.
     Dr. Eckard Wimmer demonstrated this by re-creating the poliovirus in 2001, followed by Dr. Craig Venter's synthesis of the bacteriophage phiX174 in 2003 and the 2005 re-creation of the 1918 flu virus by Dr. Jeffrey Taubenberger and Dr. Terrence Tumpey.

Dr. Craig Venter

     In Greek and Roman mythology, the chimera combined certain parts of lion, goat and serpent into one monstrous form. In the late medieval age, artists often used the creature as a symbol to illustrate the complex nature of evil. Today, a chimeric organism is a life form which contains genes from a foreign species but the goal or the end result is not always 'evil'.

     But throughout human history, abuse of science almost always occurs. Geneticists have discovered the means to increase the lethality of such biological weapons as smallpox and anthrax by tweaking their genetic structure. By combining genes, however, Scientists have discussed creating a virus that could trigger two diseases at once.
     During the late 1980s, the Soviet Union's Chimera Project, according to former Soviet researcher, Ken Alibek (see post: From Animal to Mankind) studied the feasibility of combining smallpox and Ebola into one super virus.

The Mythological Chimera

     Other potential nightmares involve scenarios of stealth viruses, strains which require certain triggers to produce infection. The stealth virus would remain dormant for a period of time until triggered by predetermined stimuli.
     Other possible chimeric weapons might require two components to become effective, such as a strain of botulinum toxin that only becomes lethal when combined with the botulinum toxin antidote.

     Before the closure of the American offensive biological warfare program by President Nixon in 1969, offensive agents had already been developed at the United States Army's biological-warfare laboratories at Fort Detrick, Maryland. These products which included powdered spores and viruses, were loaded into bombs and other delivery systems stored at Pine Bluff, Arkansas. The 1969 budget for Chemical/Biological Warfare research was reported to be $300 million with $5 million for herbicides designed to kill food crops or strip trees of foliage to deprive enemy forces of ground cover.
     By the late 1960s, the United States had developed a biological arsenal which included numerous bacterial pathogens, toxins, and fungal plant pathogens that could be directed against crops to induce crop failure and famine.

The Korean War

     During the Korean War, the Soviet Union, China, and North Korea accused the United States of waging biological warfare against North Korea and China. However, no epidemiologic evidence was ever found to support the North Korean claim of having experienced epidemics.

     Numerous other unsubstantiated allegations were made during the Cold War, including a Soviet accusation that the United States had tested biological weapons against Canadian Eskimos, resulting in a plague epidemic, an accusation that the United States had planned to initiate a cholera epidemic in southeastern China as well as another Soviet accusation of a United States and Columbian biological attack on Columbian and Bolivian peasants.

     In anticipation of the 1972 Biological Weapons Convention, President Nixon terminated the United States offensive biological weapons program by executive order in 1969. From that time on, research efforts were directed exclusively to the development of defensive measures such as diagnostic tests, vaccines, and therapies for potential biological weapons threats. Stocks of pathogens and the entire biological arsenal were destroyed but small quantities of some pathogens were retained at Fort Detrick to test the efficacy of investigational preventive measures and therapies.

     The Cobra Event is a 1998 novel by Richard Preston in which a virus, called 'cobra' was created, mixing the incurable common cold virus with one of the smallpox virus. In this fictional story, the disease that resulted from the virus was called brainpox, a genetically-engineered recombinant virus which caused nightmares, fever, chills, runny nose, encephalitis (brain swelling), and herpes-like boils in the mouth and genitals.

     But 'brainpox' may not be that far from reality. Venezuelan equine encephalitis (VEE) is an alphavirus related to a number of different viral diseases including eastern equine encephalitis and western equine encephalitis. These viruses can be easily produced in large amounts and aerosolized (for biological weapons purposes).

     Considered as a biological agent, VEE viruses are relatively stable, and can potentially injury thousands. Most importantly, the VEE virus is susceptible to genetic manipulations, which may enhance its infectiousness and virulence for biological weapons purposes. VEE is a particularly appealing biological weapon since it only requires 10-100 pathogens to infect a person. Infection with VEE infections is rarely fatal but can cause severe symptoms similar to influenza and hence can be difficult to diagnose. These encephalitis fevers cause inflammation of the brain and long-term side effects such as nervous system damage. A potential biological attack using VEE virus could be through the aerosolized route, but would be most effective during periods when mosquitoes are most active.

Venezuelan Horse Deaths in Florida

     In April 2009, 3 vials of VEE were found to be missing from the Fort Detrick research facility and soon after, 21 thoroughbred horses from a Venezuelan polo team competing in Florida suddenly dropped dead.

     The United States weaponized VEE as an offensive incapacitating agent before the termination of its biological weapons program in 1969 and the Soviet Union also harnessed VEE as a biological weapon.

     According to Ken Alibek (see post: Biological Weapons in the Twentieth Century), Soviet scientists at the All-Union Scientific-Research Institute of Molecular Biology in Koltsovo, Siberia experimented with splicing VEE genome into smallpox viruses, creating a recombinant smallpox-VEE chimera virus that resembled smallpox under a microscope but produced different symptoms in its hosts. In 1959, a freeze-dried vile containing this altered VEE was accidentally dropped by Soviet medical personnel in a stairwell and infected 20 laboratory staff.

     A report in 1998, claimed that the chimeral VEE-smallpox virus did indeed exist and that it produced a smallpox-like illness but with brain symptoms.

     In of 1998, another report claimed that the apartheid government of South Africa had initiated research in order to develop a genetically engineered biological weapon, specifically targeting blacks.
     Theoretically, it is possible to genetically engineer a virus or toxin-synthesizing gene in a bacterium which can be 'activated' by a specific gene or activated by binding to a specific receptor that determines a certain 'ethnic' characteristic (such as skin pigment or eye color). Similar genetic alterations have been achieved in cancer treatment where a unique marker (antigen) on a cancer cell is targeted by an engineered antibody against it, destroying that cancer cell.

Olfactory Neve Fibers at the Cribiform Plate

     Naegleria fowleri is a free-living type of  of protist (animal-like unicellular protozoan) typically found in warm bodies of fresh water (ponds, lakes, rivers, hot springs) but also in soil, near warm-water discharges of industrial plants, and unchlorinated swimming pools. The organism is an amoeba. Amoebae are known to cause gastrointestinal and liver disease and secondarily can affect the nervous system but what is particular about N. fowleri is that it can invade and attack the human nervous system directly. In the natural environment, although nervous system infection with this organism is rare, when it does occur, the fatality rate is close to 100%.

      Human disease caused by this protist was first described in Australia in 1965. Since 1965, more than 144 cases have been confirmed in a variety of countries.

Ameba

     N. fowleri invades the central nervous system through the nose. The organism penetrates the lining of the nasal passages, resulting in tissue death and hemorrhage. The amoeba then climbs along nerve fibers through the floor of the skull and into the brain. It  then begins to consume the brain cells piecemeal by means of a unique sucking apparatus extended from its cell surface. This results in amoebic meningoencephalitis with a victim survival rate of  less than 1%.

     Delayed diagnoses are a very significant problem to successful treatment of infection, most cases being diagnosed only after death. Infection killed 121 people in the United States from 1937 through 2007, including six in 2007  alone.

     Symptoms of infection usually present about five days after exposure and may include alteration in taste and smell, headache, fever, nausea, vomiting, and stiff neck. The next phase of symptoms consists of confusion, hallucinations, lack of attention, loss of balance, and seizures. The disease progresses rapidly over three to seven days, death occurring from 7- 14 days after exposure.

Route to Infection for
Amoebic Meningoencephalitis

     Of the 133 documented deaths in the US from this infection, 30 people were infected by contaminated recreational water and two people were infected by water from a geothermal (naturally hot) drinking water supply.

     In 2012, in Karachi, Pakistan, the nation's largest city, 10 people died of the 'brain- eating amoeba', allegedly through exposure to contaminated drinking water or bathing water.
     The source of contaminated water was unknown but it was believed that the victims may have picked it up when cleaning out their nostrils - a practice which is common in South Asia. It was suggested that people use boiled or chlorinated water to rinse out their noses and to clean out domestic water tanks which may have become contaminated by the amoeba.

     Vaccines have been developed which protect mice (and presumably humans) from N.fowleri infection. There have been no known instances of the use of this protist infection as a biological weapon. However, there have been numerous articles suggesting that this organism has a huge potential (even if not genetically altered) as an agent which can be used to contaminate water supplies and cause huge damage to enemy military and enemy populations.

     Genetic alteration of N. fowleri, of VEE or of any number of already deadly diseases could easily create an unstoppable epidemic, a disease which targets a particular population but also a disease which can easily 'jump the barrier' (in the same way as animal diseases 'jump the barrier' and affect mankind).

     There is always the risk, however, that the 'chimeral organism' could turn and affect the ones who had taken that step in 'changing the recipe'.

     * Genetic Engineering and Biological Weapons: subject of research for the novel Vaccine - Amazon Kindle.