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Genetic engineering superviruses

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.

Mae-Wan Ho


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, NC told the annual meeting of the American Association for the Advancement of Science in February [1]. The technology holds much promise, he said, 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 working 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 hijacking 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 the creation of life is that although sequencing genomes billions of basepairs in length is relatively easy, it is difficult to make DNA in the test-tube much bigger than a few thousand basepairs. 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, i.e., 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 sequences (replicated in the bacterial host) are not representative of 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.

Creation of a new 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 gastroenteritis and neurological illnesses such as multiple sclerosis. These viruses contain an 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 one of the genes of the virus is sufficient to determine its pathogenicity, 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 13 January issue of the New Scientist [4] was on a deadly virus created accidentally by researchers in Australia who were trying to genetically 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 work published in the Journal of Virology [5] revealed that the mice used were genetically resistant to the mousepox virus in the first place.

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 expression of IL-4 suppressed both NK and CTL.

Genetically resistant mice infected with the IL-4-expressing virus developed symptoms of acute mousepox accompanied by 100% mortality, similar to the disease seen when genetically sensitive mice are infected with the virulent Moscow strain. Strikingly, infection of genetically resistant mice recently immunised against mousepox also resulted in significant mortality. These findings suggest that virus-encoded IL-4 not only suppresses primary antiviral immune responses but also inhibits the expression of immune memory responses.

In previous investigations [6, 7], the IL-4 gene inserted into another virus used in vaccinations against smallpox, the vaccinia virus, delayed the clearance of the virus from experimental animals and undermined the animals’ anti-viral defence. These results suggest that IL-4 may function similarly in all viruses in the same family, which also contains the human smallpox virus.

‘More virulent than nature’s worst’

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 favouring the creation 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, however, 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 declared a moratorium in Asilomar in the mid-1970s 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 a 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, fertiliser 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 called a halt to all releases of GMOs and make sure that further research takes place only under strictly contained conditions.

Endnotes

1.   ‘Making life from scratch is now ‘imminent’: From minimal genomes: Viruses the size of HIV are likely to come firstŐ. Margaret Munro, National Post Wednesday, 21 February 2001 EDITION National Discovery PAGE A15 SAN FRANCISCO.

2.   Lai MMC. ‘The making of infectious viral RNA: No size limit in sight’. PNAS 2000: 97: 5025-7.

3.   Almazan F, Gonzalez JM, Penzes Z, Izeta A , Calvo E, Plana-Duran J and Enjuanes L. ‘Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome’. PNAS 2000: 97: 5516-21.

4.   Nowak R. ‘Disaster in the making’. New Scientist 2001: 13 Jan: 4-5.

5.   Jackson RJ, Ramsay AJ, Christensen CD, Beaton S, Diana F, Hall DF and Ramshaw IA. ‘Expression of Mouse Interleukin-4 by a Recombinant Ectromelia Virus Suppresses Cytolytic Lymphocyte Responses and Overcomes Genetic Resistance to Mousepox’. Journal of Virology: 2001: 75: 1205-1210.

6.   Bembridge GP, Lopez JA, Cook R, Melero JA and Taylor G. ‘Recombinant Vaccinia virus coexpressing the F protein of respiratory syncytil virus (RSV) and interleukin-4 (IL-4) does not inhibit the development of RSV-specific memory cytotoxic T lymphocytes, whereas priming is dimished in the presence of high levels of IL-2 or gamma interferon’. Journal of Virology: 1998: 72: 4080-7.

7.   van den Broek M, Bachmann MF, Kohler G, Barner M, Escher R, Zinkernagel R and Kopf M. ‘IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-g and nitric oxide synthetase 2’. The Journal of Immunology: 2000: 164: 371-8.

8.   ‘The genie is out’. New Scientist editorial 2001: 13 Jan: 3.

9.   Ho MW, Traavik T, Olsvik R, Tappeser B, Howard V, von Weizsacker C and McGavin G. ‘Gene Technology and Gene Ecology of Infectious Diseases’. Microbial Ecology in Health and Disease 1998: 10: 33-59.

10. ‘Dangerous GM wastes recycled as food, feed and fertilizer’. ISIS News 6, September 2000.

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New viruses and bacteria from AIDS vaccines

THERE have been intensive efforts dedicated to developing a vaccine for

AIDS, particularly in view of the raging AIDS pandemic worldwide. 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,Yugoslavia, has been warning his fellow scientists against using 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 envelope glycoprotein gene.

It turns out that the envelope 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 sequences found in bacteria and viruses such as Haemophilus influenzae, Mycobacterium tuberculosis, hepatitis B virus and herpes simplex virus that often co-infect with 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), followed by DNA sequencing, Dr Veljkovic’s group, in collaboration with groups in the UK, Italy and the 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 this point with a gene from the bacterium, Haemophilus influenzae.

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.

They 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) - MWH

Endnotes

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 development of AIDS vaccine. Vaccine 1999: 17: 1462-7.

2.   Nicolson GL, Nicolosn NL and Nasralla M.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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