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Mitchel Cohen <[log in to unmask]>
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Wed, 12 Feb 2020 12:15:15 -0500
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Relevant to today's CoronaVirus situation:

 From 2003

The Institute of Science in Society
Science Society Sustainability
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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 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, news service.
Ksiazeh TC, Erdman D, Goldsmith C et al. A novel 
coronavirus associated with severe acute 
respiratory syndrome. NEJM online 10 April, 2003.
Drosten C, Gunther S, Preiser W et al. 
Identification of a novel coronavirus in patients 
with acute respiratory syndrome. NEJM online 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, 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

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