FYI...New Technique Tracks Viral Infections, Aids Development of Antiviral Drugs

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http://www.sciencedaily.com/releases/2011/04/110411103719.htm

New Technique Tracks Viral Infections, Aids Development of Antiviral Drugs

ScienceDaily (Apr. 11, 2011) — Scientists at the Naval Research Laboratory

Center for Bio-Molecular Science and Engineering have developed a method to

detect the presence of viruses in cells and to study their growth. Targeting a

virus that has ribonucleic acid (RNA) as its genetic makeup, the new technique

referred to as locked nucleic acid (LNA) flow cytometry-fluorescence in situ

hybridization (flow-FISH), involves the binding of an LNA probe to viral RNA.

While individual parts of the technique have been developed previously, Drs.

on and Eddie Chang, in collaboration with researchers at the NRL

Lab for Biosensors and Biomaterials, demonstrate for the first time that the

combination of LNA probes with flow-FISH can be used to quantify viral RNA in

infected cells. This also allows the scientists to monitor the changes in viral

RNA accompanying antiviral drug treatment.

Once the probe is bound to the viral RNA inside mammalian cells, it is tagged

with a fluorescent dye, then thousands of these tagged cells are measured

rapidly by " flow cytometry " -- a method for counting and examining microscopic

particles, such as cells and chromosomes, by suspending them in a stream of

fluid and passing them by an electronic detection apparatus.

" The ability to rapidly measure thousands of cells for the presence of virus,

sets this technique apart from currently used methods to monitor viral

replication, " said on.

Traditionally, antibodies used to detect viruses must be produced and calibrated

for each specific strain and are highly susceptible to viral mutations. Assays

commonly used for quantifying viral loads and for drug development can be time

consuming and rely on visible signs of cell damage, which is not produced in all

viruses and can take long periods of time to occur.

Techniques such as quantitative reverse transcription-polymerase chain reaction

(qRT-PCR), microarrays, and enzyme-linked immunosorbent assays (ELISAs), while

highly sensitive, involve the lysis [the breaking down] of cells prior to

measurement and are therefore unable to provide information about cellular

viability, infected cell phenotypes, percentage of infected cells or the

variation in infection among a cell population. The LNA probe differs from

traditional nucleotide probes by binding more tightly to its target RNA.

LNA-flow FISH presents a fast and easy way to screen for compounds with

antiviral activity and could be adapted for monitoring infections in the blood

for vaccine therapy and development. This method adds a necessary tool for

several emerging areas in cell biology that enables the use of high throughput

measurements for entire populations and improves statistical analyses.

" This method can be expanded by adding more than one kind of LNA probe to enable

multiple detection of different viral and host RNA, " adds on. " The

multiplexing enhancement can be used to better understand infectious agents,

allowing this technique to be used to aid in the development of antiviral drugs

for a variety of viruses. "

LNA flow-FISH offers an advantage over other techniques due to its simplicity

and superiority. Methods involving genetic recombination of the virus to express

a fluorescent protein as a means to mark the presence of virus can utilize flow

cytometry for large-batch analysis of infected cells. However, an exception to

this approach is viral strains that have not acquired genetic mutations, known

as wild-type viruses (such as strains of Human Immunodeficiency Virus-HIV),

which would require a large initial investment of labor for engineering each

virus of interest.

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Story Source:

The above story is reprinted (with editorial adaptations by ScienceDaily staff)

from materials provided by Naval Research Laboratory