[ Superbugs and Superviruses from AIDS Vaccines below ]
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 chromosomes (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 responsible 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 exploitation 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.
Yugoslav
virologist Dr. Veljko Veljkovic warns against AIDS vaccines that can generate
new viral and bacterial pathogens and trigger cancer, on account of a
recombination hotspot in the AIDS viral gene. He intends to campaign against GM
crops with the CaMV 35S promoter for the same reasons. Dr. Mae-Wan Ho reports on
the similarity between recombination hotspots of many genes, including the prion
protein associated with mad cow disease, and their potential to generate
superviruses and superbugs.
Intensive efforts have been dedicated to developing a vaccine for AIDS,
particularly in view of the raging AIDS pandemic world-wide. But there has been
no success so far. On the contrary, Dr. Veljko Veljkovic, virologist in the
Laboratory for Multidisciplinary Research, Institute of Nuclear Sciences in
Belgrade, has been warning his fellow scientists against many AIDS vaccines
since 1990.
The vaccines on his hit-list include whole killed or attenuated AIDS
virus, HIV-1, or SIV (the related virus from monkey), recombinant HIV proteins
and peptides, recombinant viral and bacterial vectors expressing HIV proteins,
and plasmids engineered with the HIV envelop glycoprotein gene.
It turns out that the envelop glycoprotein, gp120, of HIV-1 is similar
to the region of human immunoglobulins involved in antigen-binding, a crucial
step in the immune response. Thus, any AIDS vaccine containing the glycoprotein
or the gene could strongly interfere with the immune system and make it more
vulnerable to the virus. And in the long term, it could accelerate disease
progression in HIV patients that do not yet have symptoms. Recombinant viruses
expressing gp120 could also be a source of potential new pathogens.
The gp120 gene contains elements that stimulate recombination or are
recombination hotspots. These elements are similar to certain ‘Chi’ (pronouned ‘kye’) sequences found in bacteria and viruses such as
Haemophilus influenzae, Mycobacterium tuberculosis, hepatitis B virus and herpes
simplex virus that often co-infect with the HIV, and are also similar to Ig
recombination elements in the human host. Recombination of HIV with
bacteria and viruses mediated by Chi sequences would generate new pathogens.
Within the human host, recombination with human genes would promote chromosomal
rearrangements and the formation of aberrant immunoglobulins leading to
inadequate immune responses. Furthermore, HIV-1 sequences integrated into the
genome also act as retrotransposons, and have the potential for a wide variety
of diverse genetic effects caused by all mobile genetic elements, especially
mutations of genes due to random insertion, some of which might trigger cancer.
Using reverse transcriptase followed by polymerase chain reaction
(RT-PCR), and then DNA sequencing, Dr. Veljkovic’s group, in collaboration
with groups in UK, Italy and US, isolated HIV-1 viral sequences carrying
the complete Chi recombination hotspot (GCTGGTGG) from the blood of three out of
11 AIDS patients. In one of the patients, recombination had occurred at the
hotspot with a gene from the bacterium, Haemophilus influenzae [1].
In fact, this is not the first time that recombination has been
identified involving the AIDS virus. The authors suggest that such recombination
may have been involved in a number of other cases reported in the literature.
For example, an unusual form of the bacterium Mycoplasm fermentans found
carrying part of the HIV-1 gene, has been implicated in the Gulf war syndrome
[2]. New subtypes of HIV-1 have arisen by recombination between HIV-1 subtypes.
The HI.V-1 subtype N, which is distinct from other known subtypes but very close
to the chimpanzee immunodeficiency virus, could have resulted from recombination
between HIV-1 and SIV [3]. They also suggest that the HIV-1 envelope gene could
interact with human oncogenes that have Chi sequences.
The researchers conclude:
“These results strongly support and reinforce our previous contention and the serious concern that AIDS vaccine candidates carrying the HIV-1 env gene on viral and bacterial vectors, could result in the generation of new pathogens with unpredictable effects on the immune system.” (p.1462)
“...despite the urgent need for preventive AIDS vaccines, it would be wise to introduce a moratorium on clinical trials until there is a serious reexamination of the current concepts for their development.” (p.1466)
Some readers may be aware of ISIS’ campaign to get GM crops with CaMV 35S promoter withdrawn from all environmental releases because the promoter has a recombination hotspot with all the potential hazards that Veljkovic's group has described (see “GM Rice Unstable”, this issue).
Dr. Veljkovic said, “As a consequence of the past war our country is heavily contaminated with different pollutants ranging from toxic organic substances to depleted uranium. For this reason we are expecting increase of frequency of different chronic diseases most of which could be consequence of the genomic instability induced by pollution.
“Introduction of a large amount of the GM soy containing an inherently recombinogenic element in such a vulnerable population represents an additional risk. I would like to point out another possible problem with CaMV promoter. We have demonstrated that the Chi recombination hot spot is responsible for in vivo recombination between HIV and bacteria H. influenzae ... On the other hand, the prion gene contains repeats of this recombination promoter. It will be interesting to see consequences of possible interaction between these two recombination hot spots (Chi and CaMV) after massive substitution of the bone-meal by the GM cattle-food.”
He also pointed out,
“There are some reported results indicating that genetically related retrotransposons among phylogenetically unrelated hosts can be transferred horizontally. The Ty1-like retroelement from soybean (SIRE-1), encoding a retroviral env-like protein, represents such an element. Taking into account this fact and the presence of a recombination hot-spot in the form of the CaMV promoter it is reasonable to expect problems with GM soy which will be imported here next month [April]”
Dr. Veljkovic said that he will start a campaign against the import.
1. Prljic J, Veljkovic N, doliana %, Colombatti A, Johnson E, Metlas R. and Veljkovic V. Identificaion of an active Chi recombinational hot spot within the HIV-1 envelope gene: consequences for develop-ment of AIDS vaccine. Vaccine 1999: 17: 1462-7.
2. Nicolson GL, Nicolson NL and Nasralla Mycoplasmal infections and fibromyalgia/ chronic fatigue illness (Gulf War Illness) associated with deployment to operation Desert Storm. Int. J. Med. 1998: 1: 80-92.
3. Simo F, Mauclere P, Roques P, Muler-Trutwin MC, Saragosti S, Georges-Courbot MC, Barre-sinoussi F and Brun-Verzinet F. Identification of a new human immunodeficiency virus type I distinct from group M and group O. Nature Med. 1998: 4: 1032-7.
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