In reverse genetics, the eight influenza gene segments are
transferred into cultured animal cells by a process known as
transfection. These eight gene segments are transferred within
small circular DNA molecules, called plasmids that are usually
maintained in bacterial cells. The animal host cells facilitate the
expression of the influenza gene segments into viral proteins and
also replicate the viral genome. Live influenza virus is
subsequently produced (also known as 'rescued') that can replicate
within the animal cell culture.
The use of reverse genetics to propagate selected influenza strains
is not a novel procedure, and has been used in laboratories around
the world (see Appendix I). The rescue of infectious influenza
virus from recombinant plasmid DNA encoding the influenza genome,
was first described in 1990 by Enami et al.. A reduced-set of
influenza gene segments were transfected into animal cells, along
with the purified proteins necessary for viral replication and a
helper virus to assist with assembly of functional virions. Since
this time, technical developments have allowed for the rescue of
infectious influenza virus using only recombinant plasmids, without
the need to transfect the purified polymerase proteins. The main
purpose of reverse genetics is that it allows for the rational
generation of influenza strains, with desired genetic
characteristics. Each of the eight viral segments can be derived
from different strains, and pieced together to produce a virus with
the desired reassortment of genes. It is also possible to alter
gene segments by site-directed mutagenesis, and thus genes that
confer pathogenicity (i.e. disease-causing ability) can be altered
to result in the 'rescue' of an apathogenic strain.
Rearrangement of the eight gene segments of the influenza A virus
(between different strains) already occurs in the natural world.
This process is called 'reassortment' (also known as 'antigenic
shift') and allows for the evolution of new influenza A strains
which may be able to evade the immune system of human hosts. In
reassortment, an influenza A strain from an avian species and one
from humans simultaneously infect an intermediate host such as the
pig. Genes can be swapped between the two strains and this gives
rise to new influenza A strains. It is by this process that the
1957 and 1968 human influenza pandemics arose. The influenza virus
can also change by mutation of the genome (viz. antigenic drift)
where subtle mutations in the gene sequence may result in new
strains that can evade the host immune system, or that have
increased pathogenicity or host range. It is thought that the 1918
influenza pandemic was brought about by a mutation in an avian
influenza strain that allowed efficient transmission of this highly
pathogenic virus in humans.
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