Relevant to today's CoronaVirus situation:
From 2003
The Institute of Science in Society
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SARS and Genetic Engineering?
The complete sequence of the SARS virus is now
available, confirming it is a new coronavirus
unrelated to any previously known. Has genetic
engineering contributed to creating it? Dr.
Mae-Wan Ho and Prof. Joe Cummins call for an investigation.
The World Health Organisation, which played the
key role in coordinating the research, formally
announced on 16 April that a new pathogen, a
member of the coronavirus family never before
seen in humans, is the cause of Severe Acute Respiratory Syndrome (SARS).
"The pace of SARS research has been astounding,"
said Dr. David Heymann, Executive Director, WHO
Communicable Diseases programmes. "Because of an
extraordinary collaboration among laboratories
from countries around the world, we now know with certainty what causes SARS."
But there is no sign that the epidemic has run
its course. By 21 April, at least 3,800 have been
infected in 25 countries with more than 200 dead.
The worst hit are China, with 1,814 infected and
79 dead, Hong Kong, 1,380 infected and 94 dead,
and Toronto, 306 infected, 14 dead.
A cluster of SARS patients in Hong Kong with
unusual symptoms has raised fears that the virus
may be mutating, making the disease more severe.
According to microbiologist Yuen Kwok-yung, at
the University of Hong Kong, the 300 patients
from a SARS hot spot, the Amoy Gardens apartment
complex, were more seriously ill than other
patients: three times as likely to suffer early
diarrhoea, twice as likely to need intensive care
and less likely to respond to a cocktail of
anti-viral drugs and steroids. Even the medical
staff infected by the Amoy Gardens patients were more seriously ill.
John Tam, a microbiologist at the Chinese
University of Hong Kong studying the gene
sequences from these and other patients suspects
a mutation leading to an altered tissue
preference of the virus, so it can attack the gut as well as the lungs.
The molecular phylogenies published 10 April in
the New England Journal of Medicine were based on
small fragments from the polymerase gene (ORF 1b)
(see Box), and have placed the SARS virus in a
separate group somewhere between groups 2 and 3.
However, antibodies to the SARS virus cross react
with FIPV, HuCV229E and TGEV, all in Group 1.
Furthermore, the SARS virus can grow in Vero
green monkey kidney cells, which no other
coronavirus can, with the exception of porcine
epidemic diarrhea virus, also in Group 1.
Coronaviruses
Coronaviruses are spherical, enveloped viruses
infecting numerous species of mammals and birds.
They contain a set of four essential structural
proteins: the membrane (M) protein, the small
envelope (E) protein, the spike (S) glycoprotein,
and the nucleocapside (N) protein. The N protein
wraps the RNA genome into a ‘nucleocapsid’ that’s
surrounded by a lipid membrane containing the S,
M, and E proteins. The M and E proteins are
essential and sufficient for viral envelope
formation. The M protein also interacts with the
N protein, presumably to assemble the
nucleocapsid into the virus. Trimers (3 subunits)
of the S protein form the characteristic spikes
that protrude from the virus membrane. The spikes
are responsible for attaching to specific host
cell receptors and for causing infected cells to fuse together.
The coronavirus genome is a an infectious,
positive-stranded RNA (a strand that’s directly
translated into protein) of about 30 kilobases,
and is the largest of all known RNA viral
genomes. The beginning two-thirds of the genome
contain two open reading frames ORFs, 1a and 1b,
coding for two polyproteins that are cleaved into
proteins that enable the virus to replicate and
to transcribe. Downstream of ORF 1b are a number
of genes that encode the structural and several non-structural proteins.
Known coronaviruses are placed in three groups
based on similarities in their genomes. Group 1
contains the porcine epidemic diarrhea virus
(PEDV), porcine transmissible gastroenteritis
virus (TGEV), canine coronavirus (CCV), feline
infectious peritonitis virus (FIPV) and human
coronovirus 229E (HuCV229E); Group 2 contains the
avian infectious bronchitis virus (AIBV) and
turkey coronavirus; while Group 3 contains the
murine hepatitis virus (MHV) bovine coronavirus
(BCV), human coronavirus OC43, rat
sialodacryoadenitis virus, and porcine hemagglutinating encephomyelitis virus.
Where does the SARS virus come from? The obvious
answer is recombination, which can readily occur
when two strains of viruses infect a cell at the
same time. But neither of the two progenitor
strains is known, says Luis Enjuanes from the
Universidad Autonoma in Madrid, Spain, one of the
world leaders in the genetic manipulation of coronaviruses.
Although parts of the sequence appeared most
similar to the bovine coronavirus (BCV) and the
avian infectious bronchitis virus (AIBV) (see
"Bio-Terrorism & SARS", this series), the rest of
the genome appear quite different.
Could genetic engineering have contributed
inadvertently to creating the SARS virus? This
point was not even considered by the expert
coronavirologists called in to help handle the
crisis, now being feted and woed by
pharmaceutical companies eager to develop vaccines.
A research team in Genomics Sciences Centre in
Vancouver, Canada, has sequenced the entire virus
and posted it online 12 April. The sequence
information should now be used to investigate the
possibility that genetic engineering may have
contributed to creating the SARS virus.
If the SARS virus has arisen through recombined
from a number of different viruses, then
different parts of it would show divergent
phylogenetic relationships. These relationships
could be obscured somewhat by the random errors
that an extensively manipulated sequence would
accumulate, as the enzymes used in genetic
manipulation, such as reverse transcriptase and
other polymerases are well-known to introduce
random errors, but the telltale signs would still
be a mosaic of conflicting phylogenetic
relationships, from which its history of
recombination may be reconstructed. This could
then be compared with the kinds of genetic
manipulations that have been carried out in the
different laboratories around the world,
preferably with the recombinants held in the laboratories.
Luis Enjuanes’ group succeeded in engineering
porcine transmissible gastroenteritis virus,
TGEV, as an infectious bacterial artificial
chromosome, a procedure that transformed the
virus from one that replicates in the cytoplasm
to effectively a new virus that replicates in the
cell nucleus. Their results also showed that the
spike protein (see Box) is sufficient to
determine its disease-causing ability, accounting
for how a pig respiratory coronavirus emerged
from the TEGV in Europe and the US in the early
1980s. This was reviewed in an earlier ISIS
report entitled, "Genetic engineering
super-viruses" (ISIS News 9/10, 2000), which gave
one of the first warnings about genetic engineering experiments like these.
The same research group has just reported
engineering the TGEV into a gene expression
vector that still caused disease, albeit in a
milder form, and is intending to develop vaccines
and even human gene therapy vectors based on the virus.
Coronaviruses have been subjected to increasing
genetic manipulation since the late 1990s, when
P.S. Masters used RNA recombination to introduce
changes into the genome of mouse hepatitis virus
(MHV). Since then, infectious cDNA clones of
transmissible TGEV, human coronavirus (HuCV),
AIBV and MHV have all been obtained.
In the latest experiment reported by Peter
Rottier’s group in University of Utrecht, The
Netherlands, recombinants were made of the feline
infectious peritonitis virus (FIPV) that causes
an invariably lethal infection in cats. The
method depends on generating an interspecies
chimeric FIPV, designated mFIPV, in which, part
of its spike protein has been substituted with
that from mouse virus, MHV, as a result, the
mFIPV infects mouse cells but not cat cells. When
synthetic RNA carrying the wild-type FIPV S gene
is introduced into mFIPV-infected cells,
recombinant viruses that have regained the wild
type FIPV S gene will be able to grow in cat
cells, and can hence be selected. So any mutant
gene downstream of the site of recombination,
between ORF 1a and ORF1b (see Box), can be
successfully introduced into the FIPV.
This method was previously used to introduce
directed mutations into MHV, and like the
experiment just described, was carried out to
determine the precise role of different genes in
causing disease. This targeted recombination is
referred to as ‘reverse genetics’, and depends on
the virus having a very narrow host range
determined by the spike protein in its coat.
Another research team headed by P. Britten based
in the Institute of Animal Health, Compton
Laboratory, in the United Kingdom, has been
manipulating AIBV, also in order to create
vectors for modifying coronavirus genomes by
targeted recombination, a project funded by the
UK Ministry of Agriculture, Fisheries and Food
and the Biotechnology and Biological Sciences
Research Council (BBSRC). The procedure involved
infecting Vero cells, a green monkey kidney cell
line with recombinant fowlpox virus (rFPV-T7) -
carrying an RNA polymerase from the T7
bacteriophage, with a promoter from the vaccinia
virus - together with AIBV, and a construct of a
defective AIBV genome in rFPV that can be
replicated in Vero cells. Recombinant
cornonaviruses with defective AIBV genomes were
recovered from the monkey cells. This is
significant because almost no natural
coronaviruses are able to replicate in Vero
cells; the researchers have created a defective
virus that can do so, when a helper virus is
present. The defective virus has the potential to
regain lost functions by recombination.
In addition to the experiments described, the
gene for the TGEV spike protein has been
engineered into and propagated in tobacco plants,
and Prodigene, a company specializing in crop
biopharmaceuticals, has produced an edible
vaccine for TGEV in maize. Information on whether
or not that product was the one being field
tested in a recent case of contamination reported
by the USDA was withheld under ‘commercial confidentiality’.
Sources & References
"Coronavirus never before seen in humans is the
cause of SARS. Unprecedented collaboration
identifies new pathogen in record time" WHO Press
Release, 16 April 2003, Geneva [log in to unmask]
BBC Radio 4 News Report, 19-21 April 2003.
"China says Sars outbreak is 10 times worse than
admitted" by John Gittings and Jame Meikle, The Guardian 21 April 2003.
"Chinese cover-up creates new sense of
insecuirity in face of Sars epidemic" by John
Gittings, The Guardian 21 April 2003.
"SARS virus is mutating, fear doctors" by Debora
MacKenzie, 16 April 2003, NewScientist.com news service.
Ksiazeh TC, Erdman D, Goldsmith C et al. A novel
coronavirus associated with severe acute
respiratory syndrome. NEJM online www.nejm.org 10 April, 2003.
Drosten C, Gunther S, Preiser W et al.
Identification of a novel coronavirus in patients
with acute respiratory syndrome. NEJM online www.nejm.org 10 April, 2003.
"Calling all coronavirologists" by Martin Enserik, Science 18 April 2003.
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. Engineering the
largest RNA virus genome as an infectious
bacterial artificial chromosome. PNAS 2000: 97: 5516-21.
Ho MW. Genetic engineering super-viruses. ISIS
News 9/10 , July 2001, ISSN: 1474-1547 (print), ISSN: 1474-1814 (online).
Sola I, Alonso S, Zúñiga S, Balasch M,
Plana-Durán J and Enjuanes L. Engineering the
transmissible gasteroenteritis virus genome as an
expression vector inducing lactogenic immunity. J. Virol. 2003, 77, 4357-69.
Masters PS. Reverse genetics of the largest RNA
viruses. Adv. Virus Res. 1999, 53, 245-64.
Haijema, B.J., Volders, H. & Rottier, P.J.M.
Switching species tropism: an effective way to
manipulate the feline coronavirus genome. Journal
of Virology 2003, 77, 4528 38.
Kuo L, Godeke GJ, Raamsman MJ, Masters PS and
Rottier PJ. Retargeting of coronavirus by
substitution of the spike glycoprotein
ectodomain: crossing the host cell species
barrier. J. Virol. 2000, 74, 1393-1406.
Evans S, Cavanagh D and Britten P. Utilizing
fowlpox virus recombinants to generate defective
RNAs of the coronavirus infectious bronchitis
virus. J. Gen. Virol. 2000, 81, 2855-65.
Tubolya T, Yub W, Baileyb A, Degrandisc S, Dub S,
Erickson L and Nagya EÂ. Immunogenicity of
porcine transmissible gastroenteritis virus spike
protein expressed in plants.Vaccine 2000, 18,
2023-8. Prodigene,
http://www8.techmall.com/techdocs/TS000215-6.html Sept 2001.
"Pharmageddon" by Mae-Wan Ho, Science in Society 2003, 17 , 23-4.
This article can be found on the I-SIS website at
http://www.i-sis.org.uk/SAGE.php
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