Is there a known correlation between the kind of manipulations desribed as the "art" of reverse genetics mentioned in these documents, and an
increased likelihood of future mutations? Check out "Detailed description of the invention" in the 2006 patent...these reassortant viruses used in
vaccines have been fiddled with in a dozen-odd different ways. Doesn't this destabilize the entire genetic matrix of the virus and allow for any
number of random introduced errors?
Consider the following:www.tetrahedron.org...
(speaking of dr. horowitz)
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 www.i-sis.org.uk...
), 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.