Ultra-deep sequencing with the Genome Sequencer FLX System may provide an insight into baseline viral resistance and tropism, possibly improving the way new drugs may be selected for HIV patients.
This was the conclusion drawn from two studies presented at the 16th Conference on Retroviruses and Opportunistic Infections (CROI) in Montreal, Canada.
Effective drug selection for combination therapy is critical to ensure patients suppress HIV for long periods and prevent progression to Aids.
Scientists from the University of Rome Tor Vergata and Virco BVBA used the Genome Sequencer FLX System at Virco's laboratory to analyse data generated by sequencing HIV-infected research samples.
The studies explored resistance to a new class of drug called integrase inhibitors and evaluated viral tropism in depth.
The ultra-deep method enabled by 454 Sequencing Systems improves sensitivity when detecting low-frequency viral variants, also known as viral quasispecies, compared with traditional population-based sequencing.
The first study analysed long-term antiretroviral users receiving a combination therapy of an integrase-inhibitor, raltegravir and optimised backbone therapy.
The investigators analysed samples at multiple time-points using traditional Sanger genotyping, in vitro phenotyping and the ultra-deep sequencing method.
The researchers found, at levels far below normal detection levels, an abundance of viral quasispecies in patients whose treatment failed, in contrast to fewer quasispecies found among patients whose treatment was successful.
They concluded that the co-presence of many different strains may favour the development of drug resistance and consequently drug failure.
The researchers also suggested that the correlation between quasispecies identified at baseline and treatment failure may be occurring via pathways that are more complex than simple drug-driven selection of the primary mutations currently identified.
Professor Perno, from Tor Vergata University, said: 'The ability of the technology of the 454 Sequencing System to identify mutants at very low levels provides new insights on how HIV may develop resistance to new drugs such as integrase-inhibitors.' In the second study, the researchers analysed isolates from HIV-infected patients to characterise viral tropism by traditional phenotyping and genotyping, as well as deep sequencing analysis.
HIV tropism refers to the preference of a patient's particular HIV virus to select one of two co-receptors for entry into the patient's CD4 immune cell, where the virus reproduces.
Determining the co-receptor that a HIV strain uses, either CCR5, CXCR4 or a combination of both, is a critical component of monitoring and treating HIV.
Dual-tropic virus types have the ability to use either or both co-receptors for cell entry.
In the research study, ten randomly selected dual-tropic isolates were analysed by V3 deep sequencing analysis with the 454 Sequencing System.
They were tested for research purposes in human immune cells for sensitivity to therapies, including the CCR5 antagonist maraviroc and the CXCR4 antagonist AMD3100.
From these research samples, they found that dual tropic viruses are mostly characterised as quasispecies with potential to use R5 and CXCR4 receptors as opposed to quasispecies with mixtures of R5 and X4 using viruses, an insight into how the HIV virus adapts in choosing a co-receptor to enter CD4 human target cells.
