Sunday, 9 December 2007

Virus Prevention and Control Part I

As part of preperation for my immunology final, I'm going to write a bit about controlling and preventing virus outbreaks.

When people think of controling and preventing viruses, the first thought that comes to mind is, of cource, vaccines. But vaccines are only part of the picture. There are also "low tech" methods of virus control that can prove to be effective. First of all, by controling the quality of food and water, many viruses, like noravirus, can be prevented from spreading. Poor food and water quality is a major vector for virus spread. Likewise, insects can be a vector for viruses; controling insect populations thus controls these viruses. An example of this is the recent attempts at controling the mosquito populations in North America to stem the spread of West Nile Virus. Other animal populations also need to be controled - wild mammals, for instance, to keep rabies from being spread. A very important factor which also needs to be controlled is the use and sharing of needles to stop HIV and Hepatitis C infections. A few cities have taken the controversial option of providing "safe injection sites" to prevent drug users from using unsafe needles. Education about the dangers of sharing needles has also been used as a way of preventing these diseases. Quarintine is another method of keeping virus infections from spreading, as can be seen in the SARS outbreaks in Toronto a few years ago. These "low tech" solutions often prove to be as effective as vaccines.

When it comes to vaccines, however, two strageties are used. The first is to use a live, attenuated, vaccine and the other is to use a killed, subunit, or recombinant vaccine. Each has its benefits and hazards.

Attenuated vaccines: These vaccines are made up of living virus particles which have been "attenuated", or mutated, so that they are no longer a health risk. This provides a long lasting immunity that is both humoral (invokes antibodies) and cell-mediated. There is a risk, however, that the live virus can mutate back into the fully hazardous form. This risk is minimized by using multiple non-revertable mutations to make the attenuated form. Nonetheless, this is still a risky method to use in people who are immunocompromised.

A good example of an attenuated vaccine in action is the Sabin polio vaccine. Using three different strains of the polio virus, the strains were serially passed (ie, passed from one host to another) through multiple cell tissue types. The resulting virus had accumulated non-revertable mutations in the 5' UTR and in the viral VP3 capsid protein. This resulted in a avirulant strain which was used very effectively as a vaccine.

Killed/Subunit/Recombinant Vaccines: These viruses are not living, mutated forms. They are whole or partial subunits of viruses that have been killed by heat or chemicals. They are not as effective as attenuated viruses because the immunity they provide is not as long lived and often only provokes a humoral response. Nevertheless, they are safer to use.

An example of this is the HPV vaccine. The vaccine is a mix of VLPs (virus-like particles) which consist of the viral L1 capsid protein. It gives immunity to HPV types 6, 11, 16 and 16. Interestingly, this vaccine has created some ethical questions surrounding vaccinations. HPV poses a health hazard to women but not to men. However, men can act as carriers. Should men get vaccinated even though it gives no personal benefit to them?

Antiviral Drugs: If vaccines arent a feasible option, though, then there's the option of antiviral drugs. Antiviral drugs are not as easy to come by as antibiotics. Viruses dont have nearly as many genes as bacteria, so there are not as many targets for the drugs to work against. Also, many potential antiviral drugs have toxic effects on human cells. Only a handful of antiviral drugs are known, most of which have been discovered in the last 25 years, but the rate of discovery is increasing.

How are these drugs found? There are multiple ways:
Plaque reduction assays: The virus is plated onto a lawn on tissue, forming viral plaques. An increasing amount of the potential drug is added to the plates and the effect on the plaque number is observed. The higher the decrease in plaques, the better the drug works. To determine how effective the drug is to use practically, however, the Selectivity Index (SI) of the drug must be calculated: SI=CC50/EC50 where
CC50= the concentration of the drug that kills 50% of the host cells (Cytotoxic concentration)
EC50 = the concentration that kills 50% of the virus (Effective concentration)
The higher the SI, the more effective the drug is.
Viral growth inhibition assays: The growth of a virus versus the concentration of a drug can also be visualized and used to determine the drugs effectiveness. One method is to use 3H-Thymine (radioactive thymine) to measure the amount of viral DNA replication that occurs when different concentrations of drug are applied. Another method is to use a fluorescant protein like GFP or another visual marker like the lux operon or Lac Z operon to visualize the actual viral particles in stitu or in animal tissues. The effect of differeing concentrations can then be visualized.
Enzyme inhibition assays: These assays determine how effective a drug is on a certain virus enzyme. The test require a certain amount of knowledge concerning the pharmacology of the drugs to be tested because they have to use the form of the drug that is metabolically active within cells. One technique commonly used is FRET (fluorescene resonance energy transfer). This involves measuring the amout of light emitted by a fluorecant dye versus the drug concentration. For example, if the drug target being tested was an HIV protease, then a molecule would be constricted consisting of a protein (that the protease cleaves) with a dye on one end and a quench molecule on the other. Under normal circumstances, the fluorescant dye would not emit light because the quench molecule would quench it. With an active HIV protease however, the protein would be cleaved and the flourescant dye would be free to emit light. If the drug is applied, however, then the protease would not work and there would be no light emitted. By determining the amout of light given off (or lack thereof) one can determine how effective the drug is against that HIV protease.

This type of assay can be easily done by automated machines in multi-well titre plates, resulting in high throughput screening of potential drugs against particular drug targets.

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