Use of a high resolution melting assay to analyze HIV diversity in HIV-infected Ugandan children.

HIV-infected infants usually have homogenous viral populations, suggesting that one or a few HIV variants initiate the infection [1]. Several factors promote rapid evolution of HIV viruses following infection, including the short half-life of HIV virions, the large number of virions in an infected individual (large viral population size), lack of proof-reading by HIV reverse transcriptase, and frequent viral recombination [2]. Children with established HIV infection generally harbor diverse viral populations [3]. The high level of viral diversity and the capacity of HIV to evolve rapidly in response to selective pressures can complicate HIV treatment [4, 5]. To date, studies of HIV diversity in HIV-infected infants and children have been limited because of the time, effort, and cost associated with analyzing sequences from individual HIV variants. We recently developed a rapid assay for analysis of HIV diversity that is based on high resolution melting (HRM) of DNA duplexes [6]. The HRM diversity assay uses the LightScanner instrument (Idaho Technology, Inc., Salt Lake City, UT) to measure the range of melting temperatures for DNA duplexes amplified from HIV. The assay provides a simple, single numeric measure of the level of genetic diversity in each region analyzed, which we refer to as the HRM score [6]. DNA duplexes with different sequences have different melting properties, and DNA heteroduplexes also have unique melting characteristics. In the HRM diversity assay, more diverse DNA populations (e.g., amplicons from more diverse HIV populations in clinical samples) melt over a broader range of temperatures, which is reflected in higher HRM scores. Our recent work demonstrates that the HRM diversity assay can detect single base mutations that are present at levels as low as 5% in plasmid mixtures, and can also detect insertions and deletions at low levels [7]. The HRM diversity assay is highly reproducible [8], and HRM scores are significantly associated with sequence-based measures of HIV diversity obtained from analysis of cloned HIV DNA [6] and next generation sequencing [9]. This assay facilitates studies of factors associated with HIV diversity and diversification in different regions of the viral genome. In a previous study, we used the HRM diversity assay to compare the level of diversity in the HIV gag region in HIV-infected Ugandan infants and their mothers [6]. That study confirmed that the viral populations in 6–8 week old HIV-infected infants were very homogeneous [6]. In a subsequent study, we used the HRM diversity assay to analyze diversity in three regions of the HIV genome (two regions in HIV gag and one region in HIV pol) in 31 HIV-infected Ugandan infants 6 weeks to 18 months of age. Higher HRM scores in all three regions were associated with older age, demonstrating diversification of the virus in these regions over time following infection. Furthermore, higher HRM scores at 6–8 weeks of age (scores above the 75th percentile) were associated with a significantly increased risk of death by 5 years of age [8]. In this report, we examined HIV diversity in the gag, pol, and env regions in 76 Ugandan children from 0.6 to 12.4 years of age who were enrolled in an observational study of antiretroviral therapy (ART). We analyzed the association of pre-treatment HRM scores with age (as a surrogate for the duration of HIV infection), the relationship between HIV diversity and treatment outcome, and the impact of exposure to non-suppressive ART on HIV diversity.