SARS and Genetic Engineering?

["Nello Margiotta" <animarg@tin.it>]

Subject: Genetic Engineering & the Origin of SARS
Date: Sat, 3 May 2003 16:09:46 -0400
 SARS and Genetic Engineering?
 by Dr Mae-Wan Ho and Professor Joe Cummins
 Insritute for Science in Society, 23 April 2003.


 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 thompsond@who.int 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.



Copyright Dr Mae-Wan Ho and Professor Joe Cummins 2003.  For fair use
only/pour usage équitable seulement .