New method for detecting and monitoring virus infections in cells
20 April 2011
Scientists at the US Naval Research Laboratory Center for
Bio-Molecular Science and Engineering have developed a new method to
detect viruses in cells and to study their growth.
Targeting viruses that have RNA, 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. A fluorescent dye attached to the LNA probe enables the
rapid analysis of virus activity using standard flow cytometry
While individual parts of the technique have been developed
previously, Drs Kelly Robertson and Eddie Chang, in collaboration
with researchers at the NRL Lab for Biosensors and Biomaterials,
have demonstrated 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 Robertson.
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 (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
"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 Robertson. "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 HIV),
which would require a large initial investment of labour for
engineering each virus of interest.