The past 25 years of increasing commercial exploitation of genetic engineering in both agriculture and medicine may have unleashed the potential for creating viruses and bacteria more virulent than nature's worst. Dr. Mae-Wan Ho calls for a halt to all further releases of GMOs.
Man-made, synthetic viruses with the ability to multiply by the millions are "very close", Clyde Hutchison of the University of North Carolina in Chapel Hill, N.C. told the annual meeting of the American Association for the Advancement of Science in February [1]. The technology holds much promise, but could also "potentially be misused". Already, researchers associated with a biotech company in Texas are believed to be making pieces of DNA big enough to generate viruses. But they are not releasing details of the work for "proprietary reasons".
Hutchison's team is trying to figure out the genetic recipe for creating a free-living organism from scratch. While that task is proving difficult, viruses are much easier, as they are not free-living organisms, but genetic parasites that depend on hi-jacking the cell's metabolism to replicate. According to Hutchison and other geneticists, it will soon be a relatively easy matter to tinker with existing micro-organisms to create new, more virulent varieties, and to recreate organisms that have lately become extinct. "In principle, one day someone could make smallpox".
One of the major hurdles to creating life is that although sequencing genomes billions of basepairs in length is relatively easy, making DNA in the test-tube much bigger than a few thousand basepairs gets much more difficult. That is because the enzymes that copy DNA, or RNA (the genetic material most usually found among viruses) are prone to errors. The errors are corrected by proof-reading mechanisms present only within the living cell.
This hurdle has prevented RNA viruses larger than a few thousand bases from being cloned, ie, isolated and replicated in the test-tube; until recently, that is [2]. In order to clone the virus, the RNA has to be reverse-transcribed, or copied into a complementary DNA (cDNA) sequence, which is then incorporated into a bacterial plasmid (a genetic parasite) for replicating in the bacterial cell. However, the enzymes that do the job, the reverse transcriptase and polymerase chain reaction (RT-PCR) are very error-prone, and some of the errors result in 'poison sequences' that make the cDNA unstable. Furthermore, very few vectors can accommodate long cDNA inserts.
The fidelity of RT-PCR can be improved, and has been with the help of high-fidelity reverse transcriptases becoming available. Even so, mistakes remain that have to be corrected. This procedure was used successfully in cloning the hepatitis C virus. Poison sequences arise probably because the bacteria have not been adapted to such foreign sequences. Bacteria also tend to selectively replicate certain viral sequences, so that cloned sequence (replicated in the bacterial host) is not representative those in their natural hosts. Poison sequences can be avoided by cloning the viral genome in shorter segments, which are joined together afterwards. This strategy was used in cloning flaviviruses. For vectors that can accommodate long cDNA inserts, bacterial artificial chromo-somes (BAC) are the answer. A BAC was indeed used to clone the 150 kbp herpes simplex DNA virus.
Last year, geneticists in Spain succeeded in cloning a coronavirus [3], the transmissible gastroenteritis virus (TEGV) that infects newborn piglets, giving 80% mortality. Coronaviruses include numerous economically and medically important viruses respons-ible for many common colds and possibly gasteroenteritis and neurological illnesses such as multiple sclerosis. These viruses contain a RNA genome of 17 -32 kb, more than twice the size of the largest conventional RNA viruses. Within the cell, the viral RNA is replicated entirely in the cytoplasm, outside the nucleus containing the cell's own genetic material.
The research team cloned the region containing the poison sequences last before inserting the whole into a BAC. The viral cDNA was placed under the control of a promoter from the cytomegalovirus (CMV) and the ends of the viral RNA were carefully engineered to match their natural sequence. This viral cDNA, cloned in E. coli bacteria, produced RNA viruses when injected into pigs. This was a surprise because the viral cDNA had to be transported into the nucleus of the pig cells, there to be transcribed into RNA and transported back to the cytoplasm before it could be replicated; something that the natural virus does not do. So, the research team had in effect created a new virus through genetic engineering.
Their results also showed that the 'spike' protein encoded by one of the genes of the virus is sufficient to determine its disease-causing ability, thus accounting for how a pig respiratory coronavirus emerged from the TEGV in Europe and the US in the early 1980s. The ease with which new viruses can arise, with or without the help of intentional genetic engineering should be a cause for great concern.
Since the dawn of genetic engineering in the 1970s, geneticists have found that the cDNA of many RNA viruses inserted into bacterial plasmids, were able to complete their life-cycles in bacteria. In fact, RNA genomes produced in the test-tube can also successfully transfect bacterial cells and complete their life-cycles [2]. Bacteria in the environment therefore provide a convenient reservoir for storing, multiplying and recombining viral genes to create new viruses.
The top news in the Jan. 13 issue of the New Scientist [4] was on a deadly virus created accidentally by researchers in Australia who were trying to genetic engineer a contraceptive vaccine for mice. They spliced a gene for the protein interleukin-4 (IL-4) into the relatively harmless mousepox virus in the hope that IL-4 would boost the immune system to make more antibodies. When the researchers injected this vaccine into mice, all the mice died. In fact, this synthetic virus was so lethal that it also killed half of all the mice that had been vaccinated against mousepox.
The mice killed were genetically resistant to the mousepox virus in the first place [5]. Genetic resistance to mousepox varies among inbred laboratory mice, and depends on natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) responses to viral infection, both of which destroy cells that have been infected with virus so as to clear the body of the virus. The researchers found that IL-4 suppressed both NK and CTL responses. So, the virally-encoded IL-4 not only suppresses primary antiviral immune responses but also inhibits immune memory responses.
In previous experiments [6, 7], the IL-4 gene was inserted into the virus used in vaccinations against smallpox, the vaccinia virus, and it delayed the clearance of the virus from experimental animals and undermined the animals' anti-viral defence. Thus, IL-4 may function similarly in all viruses in the same family, which also contains the human smallpox virus.
These findings raise the spectre of biological warfare. But the far greater danger lies in the unintentional creation of deadly pathogens in the course of apparently innocent genetic engineering experiments.
Genetic engineering involves facilitating horizontal transfer and rampant recombination of genetic material across species barriers, precisely the conditions favoring the creating of new viruses and bacteria that cause diseases. We now know of cases in the laboratory where such viruses have been created. But what of other viruses we know nothing about, that may have been created over the past 25 years of increasing commercial exploitation of genetic engineering in both agriculture and medicine? Genetic engineering uses the same tools and makes similar constructs, whether in agriculture or in medicine; and therefore carries the same risks.
The accompanying New Scientist editorial [8] remarked that five years ago, when biomedical researchers were asked if genetic engineering could create "a virus or bacteria more virulent than nature's worst", they replied it would be "difficult if not impossible". Some of us have been warning of 'accidents' such as this for at least the past six years. We published a detailed review on the evidence suggesting links between genetic engineering and the recent resurgence of drug and antibiotic resistant infectious diseases in 1998 [9]. We were by no means the first. Scientists who pioneered genetic engineering in the mid-1970s declared a moratorium precisely because they were concerned about this dire possibility.
Unfortunately, overwhelming pressures for commercial exploit-ation cut the moratorium short. The scientists set up guidelines based largely on assumptions, all of which have fallen by the wayside as the result of new scientific findings. Instead of tightening the guidelines, our regulators have relaxed them as commercial pressures built up. Transgenic wastes are even being recycled as food, feed, fertilizer and landfills under the current EC Directive on Contained Use [10].
Genetic engineering may have unleashed an uncontrollable, self-amplifying process of horizontal gene transfer and recombination that can sweep across the whole of the living world, with the potential indeed, of creating viruses and bacteria more virulent than nature's worst. It is time we call a halt to all releases of GMOs and to make sure that further research takes place only under strictly contained conditions.