Berkeley - Disabling a set of genes in a strain of the tuberculosis bacteria surprisingly led to a mutant form of the pathogen that multiplied more quickly and was more lethal than its natural counterpart, according to a new study led by researchers at the University of California, Berkeley.
As early as two weeks after infection, researchers found significantly more bacteria from the organs of mice infected with the mutated tuberculosis (TB) bacteria than for mice infected with the unmodified, or "wild-type," strain. By 27 weeks, the mutant-infected mice started to die, while their counterparts infected with the wild-type strain survived until the end of the experiment at 41 weeks.
"These findings came as a complete surprise to us," said Dr. Lee Riley, professor of epidemiology and infectious diseases at UC Berkeley's School of Public Health and principal investigator of the study. "We thought we had made a mistake, so we repeated the test several times, and we always got the same result."
The researchers say the study, to be published Dec. 8 in Proceedings of the National Academy of Sciences, sheds light on the mechanisms used by a pathogen that now infects one-third of the world's population and kills 2 million people per year. According to the World Health Organization, which in 1993 declared TB a global emergency, an estimated 36 million people could die of TB by 2020 if the disease is not controlled.
The results were unexpected because prior studies pointed to the mce1 operon, the collection of genes that researchers disabled in the TB bacteria, as an important virulence factor that helped the organism invade cells. Researchers expected that mutating the mce1 genes would impair the pathogen's ability to infect the mice. Instead, the bacteria became more deadly.
"This is one of the very few hypervirulent organisms ever created," said Lisa Morici, a lead author of the study who received her Ph.D. in infectious diseases from UC Berkeley in May. "This breaks a long-standing assumption among scientists that disabling a potential virulence gene weakens a pathogen."
This is the second incident that I'm aware of where investigators were trying to bioengineer a less dangerous strain of a pathogen and instead inadvertently created a far more dangerous strain. The previous case, which occurred in January 2001, was created by the addition of the gene for IL-4 (interleukin-4 which is involved in immune response) to a mousepox virus. The result was a 100% fatal mousepox.
The mousepox discovery probably presents the greater potential danger if the gene IL-4 gene, engineered into human smallpox, would have the same effect. The reason for the greater danger from the smallpox is that it is a virus and it is very difficult to develop drugs that will stop viruses. The most effective treatments for viruses to date are vaccines. If a bioengineered smallpox was essentially able to defeat the immune system then vaccines might turn out to be worthless as a means to protect against it.
This is not to say that a bioengineered TB would not be dangerous. Such a form of TB, released into a population, would kill a lot of people. But at least we'd have a fighting chance of coming up with antibiotics to save people who would be infected by it.
The other factor here is transmissibility. Would a bioengineered TB or smallpox be more transmissible? The TB might kill the victims so quickly that the victims would not be able to spread it very well. The genes that the scientists disabled may well have been selected for precisely to allow the TB to infect without killing so that the carrier could live long enough to transmit the disease to others. However, that might make it attractive for bioterrorists. If they can be assured that the release of such a pathogen would kill few beyond those infected in the initial release then the attraction would be that it wouldn't eventually spread back to their own countries.
Would measures against transmission be easier or harder to implement against the TB or smallpox? Can anyone provide an educated guess?
Surely more such accidental discoveries are in store. What we need are more discoveries, accidental or otherwise, that point the way toward how to develop better defenses against both bioengineered and naturally occuring pathogens.
A panel of life science experts convened for the Strategic Assessments Group by the National Academy of Sciences concluded that advances in biotechnology, coupled with the difficulty in detecting nefarious biological activity, have the potential to create a much more dangerous biological warfare (BW) threat. The panel noted:
- The effects of some of these engineered biological agents could be worse than any disease known to man.
- The genomic revolution is pushing biotechnology into an explosive growth phase. Panelists asserted that the resulting wave front of knowledge will evolve rapidly and be so broad, complex, and widely available to the public that traditional intelligence means for monitoring WMD development could prove inadequate to deal with the threat from these advanced biological weapons.
- Detection of related activities, particularly the development of novel bioengineered pathogens, will depend increasingly on more specific human intelligence and, argued panelists, will necessitate a closer - and perhaps qualitatively different - working relationship between the intelligence and biological sciences communities.
The Threat From Advanced BW
In the last several decades, the world has witnessed a knowledge explosion in the life sciences based on an understanding of genes and how they work. According to panel members, practical applications of this new and burgeoning knowledge base will accelerate dramatically and unpredictably:
- As one expert remarked: "In the life sciences, we now are where information technology was in the 1960s; more than any other science, it will revolutionize the 21 st century."
Growing understanding of the complex biochemical pathways that underlie life processes has the potential to enable a class of new, more virulent biological agents engineered to attack distinct biochemical pathways and elicit specific effects, claimed panel members. The same science that may cure some of our worst diseases could be used to create the world's most frightening weapons.
The know-how to develop some of these weapons already exists. For example:
- Australian researchers recently inadvertently showed that the virulence of mousepox virus can be significantly enhanced by the incorporation of a standard immunoregulator gene, a technique that could be applied to other naturally occurring pathogens such as anthrax or smallpox, greatly increasing their lethality.
- Indeed, other biologists have synthesized a key smallpox viral protein and shown its effectiveness in blocking critical aspects of the human immune response.
- A team of biologists recently created a polio virus in vitro from scratch.
According to the scientists convened, other classes of unconventional pathogens that may arise over the next decade and beyond include binary BW agents that only become effective when two components are combined (a particularly insidious example would be a mild pathogen that when combined with its antidote becomes virulent); "designer" BW agents created to be antibiotic resistant or to evade an immune response; weaponized gene therapy vectors that effect permanent change in the victim's genetic makeup; or a irstealthll virus, which could lie dormant inside the victim for an extended period before being triggered. For example, one panelist cited the possibility of a stealth virus attack that could cripple a large portion of people in their forties with severe arthritis, concealing its hostile origin and leaving a country with massive health and economic problems.
According to experts, the biotechnology underlying the development of advanced biological agents is likely to advance very rapidly, causing a diverse and elusive threat spectrum. The resulting diversity of new BW agents could enable such a broad range of attack scenarios that it would be virtually impossible to anticipate and defend against, they say. As a result, there could be a considerable lag time in developing effective biodefense measures.
However, effective countermeasures, once developed, could be leveraged against a range of BW agents, asserted attendees, citing current research aimed at developing protocols for augmenting common elements of the body's response to disease, rather than treating individual diseases. Such treatments could strengthen our defense against attacks by ABW agents.
They cited the pace, breadth, and volume of the evolving bioscience knowledge base, coupled with its dual-use nature and the fact that most is publicly available via electronic means and very hard to track, as the driving forces for enhanced cooperation. Most panelists agreed that the US life sciences research community was more or less "over its Vietnam-era distrust" of the national security establishment and would be open to more collaboration.
- One possibility, they argued, might be early government assistance to life sciences community efforts to develop its own "standards and norms" intended to differentiate between "legitimate" and "illegitimate" research, efforts recently initiated by the US biological sciences community.
- A more comprehensive vision articulated by one panelist was for the bioscience community at large to aid the government by acting as "a living sensor web" - at international conferences, in university labs, and through informal networks - to identify and alert it to new technical advances with weaponization potential. The workshop did not discuss the legal or regulatory implications of any such changes.
Attempts to prevent the spread of nuclear weapons technology are already glaringly inadequate and are failing (and also see here). Efforts to prevent bioweapons development will fare even worse because the "footprint" of a bioweapons development effort will be able to be incredibly smaller than that of a nuclear weapons development effort. The development of microfluidics devices and nanotechnology hold the potential to revolutionize medical research, disease treatment, and the development of rejuvenation therapies. But those same technologies also will make it easier to use biotech for nefarious purposes.
Illustrating the speed with which biotechnology is advancing to create new bioterrorism threats is a recent announcement by Craig Venter and his Institute for Biological Energy Alternatives that they have synthetically created working copies of the known existing bacteriophage virus Phi X174.
Scientists at the Institute for Biological Energy Alternatives (IBEA) in Rockville, Maryland , announced their findings, along with the Secretary of the Department of Energy (DOE), Spencer Abraham, at a press conference Thursday in Washington, D.C. DOE funded the research.
J. Craig Venter, president of IBEA, led the research, working with longtime collaborators Nobel Laureate Hamilton O. Smith of IBEA and Clyde A. Hutchinson of the University of North Carolina, Chapel Hill. Venter and Smith were principal collaborators on sequencing the human genome. Smith, in his 70s, and Hutchinson, in his 60s, pulled all-nighters “just like post-docs” to create the genome in record time, said Venter.
Venter and his colleagues created the genome of a virus that infects bacteria but is harmless to humans. The genome of this particular virus, called phi X, was already a bit of a celebrity in the world of genomics. In 1978, it was the first virus ever sequenced. It has been extensively studied in the laboratory since the 1950s.
In 14 days, the researchers created the artificial phi X by piecing together synthetic DNA ordered from a biotechnology company. They used a technique called polymerase cycle assembly (PCA) to link the strands of DNA together.
As a demonstration, researchers at the Institute for Biological Energy Alternatives announced yesterday that they had created a simple virus in just 14 days by stitching together strands of synthetic DNA purchased through the mail.
"You can envision this like building something out of Legos," said IBEA President J. Craig Venter, who led the race to decode human DNA before joining the effort to build organisms from scratch.
The team used enzymes to glue the oligonucleotides together accurately into the complete 5,386-base genetic strand, and to copy it many times. When the synthetic viral genome was injected into bacteria, the bacterial cell's machinery read the instructions and created fully fledged viruses.
By contrast, the previously synthesized 7500 base long poliovirus synthesis project took two years and the resulting poliovirus had errors in its DNA sequence.
Other researchers had previously synthesised the poliovirus, which is slightly bigger, employing enzymes usually found in cells. But this effort took years to achieve and produced viruses with defects in their code.
So the timescale has shifted from years to weekst o make a virus. There are other bigger viruses that would require more time to assemble. The biggest viruses are 400,000 base pairs long with HIV containing 10,000 base pairs whereas hepatitis B contains 3000, human cytomegalovirus contains about 230 kilo base pairs (kbps where kilo means thousand) and influenza at 12 kbp. By contrast the E Coli bacteria is 4 million base pairs, the the bacteria that causes tuberculosis is 4,411,532 base pairs (bp) and the bacteria that causes leprosy is 3,268,203 bp. So building artificial bacteria from scratch is a much bigger job. But keep in mind that 12 kbp number for influenza. Individual influenza strains have killed tens of millions of people. Imagine a bioengineered influenza attack that unleashed many deadly strains at once. The results for the human race would be catastrophic.
Before the work was publicized, officials at the Department of Energy consulted with the White House and the Department of Homeland Security to make sure there were no security concerns. And the paper describing the results, which will be published in the Proceedings of the National Academy of Sciences, was subjected to an extra level of scientific review, according to Venter, who heads the Institute for Biological Energy Alternatives in Rockville, Md., where the work was done.
Note from the previous article that Venter thinks his team could make a bacteria with about 60 times larger genome from scratch within about a year of starting.
The Venter team used improvements of a process called polymerase cycling assembly (PCA) to achieve the fast construction time. This technique could also be applied to dangerous viruses whose sequences are known.
But the DNA sequences of several nasty viruses, including smallpox, are now known and publicly available. And as one of the team observed, the entry proteins for smallpox might be provided by a related but harmless virus. Let’s hope nobody tries.
The debate about whether to destroy smallpox virus stocks is pointless because any virus or bacteria whose DNA sequence is published is eventually going to be easily creatable by labs all around the world.
Researchers are investigating how to counter the danger posed by the IL-4 gene when inserted into mousepox virus. (also same article here)
SAN FRANCISCO — A research team backed by a U.S. federal grant has created a genetically engineered mousepox virus designed to evade vaccines, underscoring biotechnology's deadly potential and stirring debate over whether such research plays into the hands of terrorists.
The team at the University of St. Louis, led by Mark Buller, created the superbug to figure out how to defeat it, a key goal of the government's anti-terrorism plan.
The fear is that terrorists may take the IL-4 gene used in this research and put it into the human smallpox virus instead and thereby perhaps make a much more lethal form of smallpox. It is possible that vaccines would not provide any protection against such a strain of smallpox. IL-4 may effectively disable the immune system and any vaccine development attempts may be futile. Even a vaccine against IL-4 probably wouldn't work because IL-4 serves a useful role in regulating human immunity.
Now Buller has engineered a mousepox strain that kills 100 per cent of vaccinated mice, even when they were also treated with the antiviral drug cidofovir. A monoclonal antibody that mops up IL-4 did save some, however.
This work replicates and extends upon work first reported back in January 2001 where some Australian CSIRO scientists were accidentally trying to develop a mouse contraceptive and produced an extremely lethal strain of mousepox instead.
Ron Jackson and Ian Ramshaw weren't looking for trouble. Jackson, who works at the Pest Animal Control Cooperative Research Centre in Canberra, and Ramshaw, who is in the same city at the Australian National University, were searching for a way to control the mice that are serious pests in Australia. They wanted to make a contraceptive vaccine by altering the genes of the mousepox virus.
Accidental architect: Ron Jackson co-engineered a particularly virulent form of mousepox. But in January the project gained notoriety after the pair inadvertently created an unusually virulent strain of mousepox. If a similar genetic manipulation were applied to smallpox, the scientists realized, this feared killer could be made even more dangerous. When they published their paper1, it was only after much discussion about the wisdom of drawing attention to the findings. "It has to be brought out into the public arena so the situation can be addressed," argues Ramshaw.
There are a few things to worry about in all this. First of all, Ramshaw's team were not even looking for a way to make mousepox more lethal. This will certainly not be the last time that researchers will accidentally find ways to make pathogens more lethal. Would-be terrorists in the future will find many methods published in the research literature for how to make a wide variety of pathogens more lethal. Just research on why some strains of influenza are more virulent than others will have potential terrorist uses.
This brings up the debate on whether all scientific research should be published in public journals. Are there types of research results that will be so incredibly dangerous that they will make it too easy for nefarious groups to harm others on a massive scale? Will it be harder to defend against such attacks than it will be to use those publically available reports to develop effective defenses? While many proponents of open societies take it as a matter of faith that more openness and availability of information is always better that seems far from a proven position.
University of Texas researchers see influenza as a greater bioweapons threat than smallpox.
The Texas researchers wrote in the Journal of the Royal Society of Medicine that scientists are close to completing the blueprint of the 1918 Spanish Flu (search) that killed 20-40 million people around the world, including half a million in the United States. That blueprint, they said, could provide the recipe for terrorists looking for a deadly weapon.
The sequencing of the 1918 Spanish Flu DNA has been underway for quite some time as this 2001 PNAS paper demonstrates. Once it is complete the sequence will be available for use by any group capable of building a virus from scratch or by any group that can modify an existing influenza virus strain to put in the genetic variations that caused the level of virulence that was characteristic of the 1918 strain.
The complete sequence of the 1918 flu will be known within 2 years. Madjid says that it will become increasingly easier to build the 1918 virus from the sequence information.
Dr Madjid told BBC News Online: "Using influenza as a bioweapon is a probability.
"It's just a matter of technology. If it's difficult now, it will be easier in six months and much easier in a year's time."
He is right about the increasing ease of creating such a virus. This is more generally true of building almost any kind of weapon you can imagine. The more technology advances the easier it becomes to make things.
Here is the Pub Med entry for the paper by Mohammad Madjid MD, Scott Lillibridge MD, Parsa Mirhaji MD, and Ward Casscells MD on Influenza as a bioweapon. From the Royal Society of Medicine Press Release on this paper.
Many bioterror warnings have focused on diseases like smallpox, but flu has very different implications for public health. Influenza is far more easily available, and common enough that a cluster of cases would not cause alarm at first. Once an epidemic has begun, it is more difficult to immunize against, as the incubation period is short. The virus is very difficult to eradicate since birds, rats and pigs all carry flu.
A third difference is that the incubation period for influenza is short (1–4 days) versus 10–14 days for smallpox. Immunization after exposure to influenza is therefore not protective, and even the neuraminidase inhibitors such as oseltamivir must be administered before symptoms develop or within the first 48 hours after their appearance. Fourth, influenza is harder to eradicate, because of avian, murine, and swine reservoirs. Fifth, influenza outside of pandemics, has lower case-fatality (2.5% versus 25%, though the newly recognized triggering of cardiovascular events suggests that the true mortality may be much higher in ill or elderly persons). Finally, influenza poses a greater threat to world leaders than does smallpox, because they are older and prone to influenza and its cardiovascular complications, have some residual immunity to smallpox (whereas unvaccinated youth have none), and are often in public places.
As I've previously argued, we need facilities that are capable of being operated in a crisis mode to very rapidly sequence and make a vaccine for a new killer strain of influenza. That strain might arise naturally (this seems inevitable in fact) or it could be made by terrorists. But eventually we are going to be faced with it. The development of DNA vaccine technology for influenza has the potential of enabling the manufacture of much greater quantities of vaccine more quickly and cheaply than conventional vaccine manufacturing approaches. Therefore DNA vaccine development for more influenza strains should be a priority as a useful learning experience.
Jeanne Kwik and others at the Johns Hopkins Center for Civilian Biodefense Strategies have written a paper examining the threat that terrorists will be able to uses advances in biotechnology to make biological weapons of mass destruction.
December 20, 2002
Researchers Warn Biotech Advances Could Be Misused By Terrorists
Center for Civilian Biodefense Strategies Urges Oversight of Scientific Information
The same scientific advances in biotechnology, genetics, and medicine that are intended to improve life could also be used to develop biological weapons capable of causing mass destruction, according to researchers from the Johns Hopkins Bloomberg School of Public Health’s Center for Civilian Biodefense Strategies. They urge governments and the scientific community to adopt a system of checks and balances to prevent the misappropriation of scientific discoveries and technology. Their analysis is outlined in an article published in the January 2003 edition of the journal Biosecurity and Bioterrorism.
The Hopkins researchers call the potential misapplication of science the “Persephone effect,” named after the Greek myth of an innocent girl who was kidnapped and forced to share her time between Hades and Earth. The myth accounts for the change of the seasons and the annual cycle of growth and decay.
“Biology, medicine, agriculture, and other life sciences were always considered the ‘good’ sciences, but like Persephone they could be used to bring death and destruction in the form of biological weapons,” explained lead author Gigi Kwik, PhD, a fellow with the Johns Hopkins Center for Civilian Biodefense Strategies and assistant scientist in the Department of Health Policy and Management at the Johns Hopkins Bloomberg School of Public Health.
According to Dr. Kwik and her colleagues, recent advances in aerosol technology, microbiology, and genetics are areas of concern. In the article, they noted that the same aerosol technology used to develop inhaled insulin for the treatment of diabetes could also be used to push anthrax or other large molecules past the lung’s immune system and deep into the airways where they can cause disease. Antibiotic-resistant strains of bacteria help scientists determine which antibiotic therapies will be most effective in treating an illness, but former Soviet bioweapons builders are suspected using this technology to develop antibiotic-resistant forms of plague, anthrax, and tularemia. Last year, Australian researchers inadvertently created a lethal form of mousepox by adding a single gene to the virus, and this year scientists in the United States were able to create polio virus from scratch by assembling pieces of DNA. The Hopkins researchers suggest these technologies could make harmless unregulated organisms dangerous and render obsolete current policies to restrict access to dangerous pathogens.
Technology is just a way of doing things. It can be used for good or ill. But we appear to be reaching a stage of technological development where it is becoming easier for relatively small groups to use technology for tremendous ill. One of the characteristics of advanced technology is that it lets us more easily do more complex manipulations of matter. Technology advances to first make a previously impossible task possible to do if one has a great deal of money and highly skilled workers. So, for instance, it look most of the best physicists on the planet and the resources of the richest nation to build the first nuclear bomb. But as technology advances further the difficulty diminishes. It takes less money, fewer people, and less skilled people to accomplish some task because more advanced technologies are available to help do it. We can see the consequence of this for nuclear bomb development where much smaller and poorer nations can build nuclear bombs using less resources and fewer and less able scientists.
One of my greatest worries for the 21st century is that technological advances will shift the battlefield in favor of effectively anonymous attackers (i.e. attackers who attempt to blend into the societies that they attack and who are rarely seen operating as fighters). Such attackers may not even choose to operate as terrorists because death rather than terror may be their main objective. This trend could run so far that civilization will be very difficult or perhaps even impossible to defend.
This threat looks set to grow larger with time. The more that biological science and biotechnology advance the easier it will be to modify pathogens to make them more lethal and to create delivery systems that are more effective at getting pathogens into humans and into agricultural plants and livestock. Technology makes things easier to do. The problem is that the ability to attack may well advance more rapidly than the ability to defend. There have been periods in history when technological advances shifted the balance in favor of the defenders (eg in the modern era machine guns contributed to the trend near the end of the US Civil War when trench warfare began and then in WWI trench warfare reached its widest application) and other periods in history when technological advances shifted the balance back in favor of the attackers (eg the maturation of the tank helped to end the era of trench warfare after WWI).
For most of the modern era even when the state of technology has favored attackers it favored large state attackers. Civilization could still organize itself around the most powerful states. But what happens if we find ourselves in a situation where it becomes incredibly easy for small groups to build devices (eg mini-nukes or bioweapons) that can cause huge amounts of devastation? Defense may become so much harder than attack that large organized polities may become extremely hard to defend. There may be no technological solution to this problem.