- 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? <mailto:m.w.ho@i-sis.org.uk>Dr.
Mae-Wan Ho and <mailto:jcummins@uwo.ca>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 ÔnucleocapsidÕ thatÕs 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 thatÕs 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 "<http://www.i-sis.org.uk/BioTerrorismAndSARS.php>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 EnjuanesÕ 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" (<http://www.i-sis.org.uk/isisnews/i-sisnews9.php>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 RottierÕs
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 Ôreverse geneticsÕ,
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 Ôcommercial
confidentialityÕ.
-
- Sources & References
-
- 1. "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 <mailto:thompsond@who.int>thompsond@who.int
BBC Radio 4 News Report, 19-21 April 2003.
- 2. "China says Sars outbreak is 10 times worse than
admitted" by John Gittings and Jame Meikle, The Guardian 21 April
2003.
- 3. "Chinese cover-up creates new sense of insecuirity
in face of Sars epidemic" by John Gittings, The Guardian 21 April
2003.
- 4. "SARS virus is mutating, fear doctors" by
Debora MacKenzie, 16 April 2003, NewScientist.com news service.
- 5. Ksiazeh TC, Erdman D, Goldsmith C et al. A novel coronavirus
associated with severe acute respiratory syndrome. NEJM online <http://www.nejm.org/>www.nejm.org
10 April, 2003.
- 6. Drosten C, Gunther S, Preiser W et al. Identification
of a novel coronavirus in patients with acute respiratory syndrome. NEJM
online <http://www.nejm.org/>www.nejm.org 10 April, 2003.
- 7. "Calling all coronavirologists" by Martin
Enserik, Science 18 April 2003.
- 8. Lai MMC. The making of infectious viral RNA: No size
limit in sight. PNAS 2000: 97: 5025-7.
- 9. 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.
- 10. Ho MW. Genetic engineering super-viruses. <http://www.i-sis.org.uk/isisnews/i-sisnews9.php>ISIS
News 9/10 , July 2001, ISSN: 1474-1547 (print), ISSN: 1474-1814 (online).
- 11. Sola I, Alonso S, Z-iga S, Balasch M, Plana-Dur¦n
J and Enjuanes L. Engineering the transmissible gasteroenteritis virus
genome as an expression vector inducing lactogenic immunity. J. Virol.
2003, 77, 4357-69.
- 12. Masters PS. Reverse genetics of the largest RNA viruses.
Adv. Virus Res. 1999, 53, 245-64.
- 13. 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 Ð 38.
- 14. 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.
- 15. 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.
- 16. Tubolya T, Yub W, Baileyb A, Degrandisc S, Dub S,
Erickson L and Nagya Eå. 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>http://www8.techmall.com
- /techdocs/TS000215-6.html Sept 2001.
- 17. "Pharmageddon" by Mae-Wan Ho, <http://www.i-sis.org.uk/isisnews/sis17.php>Science
in Society 2003, 17 , 23-4.
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