ISIS Report 06/03/01
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. 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 . 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
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
are genetic parasites that depend on hi-jacking the cells 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, ie, isolated and replicated in the test-tube; until
recently, that is . 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
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 have succeeded in cloning a coronavirus
, 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 cells own
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 . 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  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 have been vaccinated against mousepox.
The work published in the Journal of Virology , 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 immunized against the mousepox
also resulted in significant mortality. These findings suggest that
virus-encoded IL-4 not only suppresses primary antiviral 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, delay 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
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  remarked that five
years ago, when biomedical researchers were asked if genetic engineering could
create "a virus or bacteria more virulent than natures 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 . 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 .
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
- 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.
- 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:
- 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:
- "The genie is out" New Scientist editorial 2001: 13 Jan.
- 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