HIV can enter cells and integrate into
the cell's genome and remain indefinitely in an inactive form. In this
integrated dormant state, even antiretroviral drugs, which attack HIV, are not
able to kill the virus.
A novel genetic technique
developed that can accurately measure the amount of the viral particles
integrated within the cell's genome, which is critical for evaluating the
efficacy of new antiretroviral therapies
The study was led by Prof. Robert F.
Siliciano, MD, PhD, who is an Investigator at the Howard Hughes Medical
Institute and a Professor of Medicine at the Johns Hopkins University School of
Medicine, Maryland, USA. He also holds joint appointments in the Departments of
Molecular Biology and Genetics, Pharmacology and Molecular Sciences, and
Biology, at the Johns Hopkins School of Arts and Sciences.
The study has been published in Nature
prestigious journal of Springer Nature Publishing, UK.
Detecting and Quantifying HIV
During the replication cycle of HIV, it
infects a population of immunological cells known as CD4+
which circulate in the blood. Following infection, HIV
continues to replicate within these CD4+
cells, resulting in the integration of the viral genes into the genome of
these cells. Therefore, the viral genetic information remains stored in a
latent state within the CD4+
cells, which make it extremely
difficult to detect, much less quantify.
Trying to measure this 'latent reservoir'
of HIV particles using conventional techniques has yielded erroneous results,
sometimes overestimating up to 100-folds. This has necessitated the development
of newer techniques that can not only detect, but also count the latent viral
New Techniques for
Detecting and Quantifying HIV
The development of the new technique occurred in three stages, over many
years of experimentation. These are highlighted below:
Viral Outgrowth Assay (QVOA): The basis of the new technique originated in
1995 when Siliciano and his team discovered the 'latent reservoir' of HIV
by using a technique called Quantitative Viral Outgrowth Assay (QVOA),
which was developed by them. However, a major drawback of this assay is
that it is very difficult to grow the HIV-infected cells, coupled with the
fact that it is very cumbersome and time-consuming, often taking several
weeks to complete the assay.
PCR Technique: In order to circumvent the above problems, researchers
prefer to use a simpler technique known as the Polymerase Chain Reaction
(PCR), which is widely used in Molecular Biology labs. PCR detects the
levels of viral DNA (deoxyribonucleic acid - the genetic code of an
organism) in the CD4+ cells. But even this technique is not
absolutely perfect for the accurate estimation of viral DNA, as 98 percent
of the DNA is harmless. Only the remaining two percent viral DNA is
harmful because it can reactivate to cause re-infection, and is thus
relevant for monitoring the health status of the patient. Therefore, the
original PCR technique also suffers from an overestimation of the viral
particles present in CD4+ cells.
PCR Technique: To overcome these drawbacks of the original PCR technique,
Siliciano's team designed a new type of PCR that is capable of distinguishing
between intact and defective segments in the viral DNA. This novel
technique uses dual-colored fluorescent probes that are capable of binding
to DNA regions that are highly prone to undergo mutations (changes),
thereby causing genetic defects in the virus. A color read-out allows for
quantification of the genetic defects in the viral DNA template.
Therefore, this new technique gives an accurate measurement of the levels
of harmful HIV particles present in the CD4+ cells. This can
help monitor the patient's response to various antiviral therapies
accurately without the risk of overestimation, which had plagued the
The new technique is a landmark
development in HIV diagnostics. Now, the efficacy of an antiretroviral drug or
a combination of drugs can be assessed to see if they are able to destroy the
HIV hiding within the cellular genome by measuring the activity of the virus.
decades, the field has been clamoring for an accurate measure for these hidden viral
says. "Now, we have a good way to know if
we are making a dent in their numbers."
He adds: "We may still be a long way from a cure,
but now at least we can measure our progress." Reference :
- A Quantitative Approach for Measuring the Reservoir of Latent HIV-1 Proviruses - (http://dx.doi.org/10.1038/s41586-019-0898-8)