Fwd: SARS and Genetic Engineering?

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SARS and Genetic Engineering?

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The Institute of Science in Society

Science Society Sustainability

http://www.i-sis.org.uk

 

General Enquiries sam

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ISIS Director m.w.ho

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SARS and Genetic Engineering?

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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 (m.w.ho) and Prof. Joe

Cummins (jcummins) 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.

 

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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.

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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 http://www.i-sis.org.uk/BioTerrorismAndSARS.php), 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 http://www.i-sis.org.uk/isisnews/i-sisnews9.php), 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 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

http://www.i-sis.org.uk/isisnews/i-sisnews9.php, 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

http://www.i-sis.org.uk/isisnews/sis17.php.

 

 

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General Enquiries sam

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