In this weeks installment we look at the mechanisms of Tamiflu and other neuraminidase inhibitors, rapid diagnostic tests, and improvements in reverse engineering of influenza viruses.
The mechanisms of action of
TamifluTamiflu's pharmaceutical name is
oseltamivir phosphate, while its chemical name is
(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylic
acid, ethyl ester, phosphate. Are you glad you know? Here is the chemical structure of oseltamivir phosphate
This compound and others are inhibitors of the neuraminidase enzyme of the
influenza virus. Neuraminidase is essential for release of the virus
from its host, with its specific activity being to hydrolyze the sialic acid end of glycoproteins and glycolipids present on the surface of host cell membranes. This activity is thought to be essential for
virus mobility, preventing aggregation of the virus and release from
host cell membranes. Oseltamivir phosphate is a sialic acid analogue,
mimicking the substrate and binding to the active site of
neuraminidase.
This prevents the enzyme from carrying out its activity. Hemagglutinin
then binds sialic acid on the surface of its host, thus trapping the
virus and preventing its spread to new cells. Oseltamivir was
developed using rational drug design, which requires a crystal
structure of the enzyme that is being targeted. Here is a crystal
structure of neuraminidase binding an inhibitor of similar structure to
oseltamivir. The protein is shown in blue, with the inhibitor show in
red and yellow. As you can see, the inhibitor binds in the active site
of the enzyme in a pocket. Once bound, the enzyme cannot attack sialic
acid.
Resistant
mutants to oseltamivir are known.
Wang et.al. in a 2002
paper describe analysis of mutants resistant to neuraminidase
inhibitors and the changes that accompany them. There were laboratory
studies where the virus was exposed to incrementally increased levels
of oseltamivir, allowing it to mutate. In a clinical situation, gradual
increases in drug concentration would be avoided. Importantly, in all
cases the mutation of neuraminidase to resistance was also accompanied
in a reduction of enzyme activity and infectivity.
A rapid assay for influenza using
RT-PCRWhiley and Sloots have
developed a rapid assay for the presence of flu virus using real time
PCR (RT-PCR) that allows the detection of virus in a matter of hours.
This method can detect all flu viruses, including H5:N1. The assay can
be developed to look for a specific viral strain, a subset of viruses,
or all influenza viruses depending upon how it is designed. Use of this
method should greatly speed detection of the virus and surveillance,
which is critically important to monitoring and stopping the virus.
Faster reverse genetics
Reverse
genetics, in the influenza field, refers to the the collection of
techniques that produces influenza viruses from cloned cDNA.
These methods are essential for the creation of modern
influenza vaccines. Established systems for the artificial generation
of influenza A viruses require transfection of cells with the eight to
12 plasmids that provide the eight influenza viral RNAs as well as the
polymerase and nucleoproteins of the virus.
Neumann et. al.report the
creation of a
simplified system, that only requires 4 plasmids, instead of 12. Under
these conditions, transfection was 100 to 1000-fold more efficient.
Since the 12 plasmid method was of low enough efficiency that it often
failed, this new method should greatly increase success rates. The last
paragraph of the article, summarizes its importance quite well.
In summary, here we present an improved system for
the generation of
influenza viruses that allows the easy and reproducible production of
vaccine viruses in Vero cells. Application of this system may be
especially advantageous in situations of outbreaks of highly pathogenic
avian influenza viruses.