GENETIC  ENGINEERING  SUPERVIRUSES  -  ISIS Report  06/03/01

The past 25 years of increasing commercial exploitation of genetic engineering in both agriculture and medicine may have unleashed the potential of 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]. He warned 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 working to figure out the genetic recipe needed to a create a free-living organism from scratch. While that task is proving difficult, viruses are much easier, as they are not free-living organisms, but are 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, 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 are prone to errors. These 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 isolated and replicated in the test-tube, until recently, that is [2]. In order to do that, the RNA has to be reverse-transcribed into a complementary DNA (cDNA) sequence, which is then incorporated into a bacterial plasmid for replicating in the bacterial cell. However, the reverse transcriptase and polymerase chain reaction (RT-PCR) required for copying and amplifying the RNA sequence into cDNA are very prone to errors, and some of the errors result in ‘poison sequences’ that make the cDNA sequence in plasmids 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 (those replicated in the bacterial host) are not representative of the population of the viral sequences in their natural hosts. Poison sequences can be minimized by cloning the complete sequence in shorter segments, which are then 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 have succeeded in cloning a coronavirus [3], 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 virus contain a positive-sense (the coding strand) RNA genome of 17 ­32 kb, more than twice the size of the largest DNA of the conventional RNA viruses. The viral RNA is replicated by an RNA-dependent RNA polymerase entirely in the cytoplasm, independently of the nucleus.

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 the cytomegalovirus (CMV) immediate-early promoter and the ends of the viral RNA were carefully engineered to match their natural sequence. This viral cDNA transfected into E. coli bacteria gave successful production of viral RNA, despite the fact that the viral cDNA had to be transcribed into RNA in the nucleus and exported into the cytoplasm before it could be replicated, something that the natural virus itself does not do. So, the research team had actually created a new virus through genetic engineering.

Their results also showed that the spike protein encoded by the virus is sufficient to determine its pathogenicity, thus explaining how a porcine 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].

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 into the relatively harmless mousepox virus in the hope that interleukin-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 have 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 effective 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 immunized against the mousepox also resulted in significant mortality. These findings suggest that virus-encoded IL-4 not only suppresses primary antiviral cell-mediated immune responses but also inhibit 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, was found to delay the clearance of the virus from experimental animals and to counteract 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. IL-4 belongs to a group of proteins, known collectively as cytokines, which regulate immune response of cells, and include interferons and tumour necrosis factors.

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 about those unseen cases that may have been going on 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 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 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.

"Making life from scratch is now ‘imminent’: From minimal genomes: Viruses the size of HIV are likely to come first" Margaret Munro, National Post Wednesday, February 21, 2001 EDITION National Discovery PAGE A15 SAN FRANCISCO.
Lai MMC. The making of infectious viral RNA: No size limit in sight. PNAS 2000: 97: 5025-7.
Almazan F, Gonsalex JM, Penzes Z, Izeta , 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.
Nowak R. Disaster in the making. New Scientist 2001: 13 Jan. 4-5.
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.
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.
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.
"The genie is out" New Scientist editorial 2001: 13 Jan. 3.
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.
"Dangerous GM wastes recycled as food, feed and fertilizer" ISIS News 6, September 2000
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