SARS and Genetic Engineering?

The Institute of Science in Society

London, England

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@i-sis.org.uk) and Prof. Joe Cummins (jcummins@uwo.ca) 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 (Click Here), 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 fun ctions by ecombination.

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 http://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 http://www.nejm.org 10 April, 2003.

"Calling all coronavirologists" by Martin Enserik, Science 18 April 2003.

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http://www.i-sis.org.uk/isisnews/i-sisnews9.php, July 2001, ISSN: 1474-1547 (print), ISSN: 1474-1814 (online).

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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.

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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.

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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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