Consideration of multiple template switches is critical to the determination of the recombination rate on the HIV genome

Retroviral recombination drives viral diversity and facilitates the emergence of immune escape and drug resistant mutants that contribute to disease progression. Current estimates of retroviral recombination rates are based on indirect measurements that do not take into account the effects of multiple recombination events. In the presence of multiple template switches, any even number of template switches result in no observed recombination and any odd number is detected as a single recombination event. We demonstrate that ignoring multiple recombination events consistently underestimates the true recombination rate, especially over large genetic distances and high rates of recombination. Here, we present a novel approach to measure rates of recombination across different gene segments regardless of the effects of genetic distance and the overall rate of recombination. We apply these tools to a novel HIV-1 marker system, which mimics the recombination process between closely related genomes, analogous to those found within the quasispecies of an infected individual. We directly measure the recombination rate in gag, correcting for the effects of multiple template switches and background recombination. Furthermore, our analysis indicates that recombination rates are likely to vary across the viral genome. This system is applicable to other studies to accurately measure the recombination rate that is critical for the diversification of retroviruses.

Introducing the D4 Diagnostic Quadrant as a targeting tool: developing a framework for the effectiveness of HIV/AIDS interventions in Tanzania and beyond

The current study develops and evaluates a tool to distinguish four different categories of educators for the effective delivery of HIV/AIDS health education using data from 548 randomly selected participants aged 16 years. The D4 Diagnostic Quadrant is based on HIV knowledge and sexual practice behaviours and indicates four distinct typologies of educator. The discerning educator has high HIV/AIDS knowledge and healthy sexual practices. The dissolute educator has high HIV/AIDS knowledge but employs unhealthy or risky sexual practices. The decorous educator has low HIV/AIDS knowledge but practices healthy sexual practices. The disempowered educator has low HIV/AIDS knowledge and employs unhealthy or risky sexual practices. The study found that the two categories that will result in the most effective behaviour-change interventions are those that target ‘discerning’ and ‘decorous’ individuals as the educators. Both these categories have underlying healthy practices that minimise the risk of HIV transmission. The D4 Diagnostic Quadrant tool provides information as to existing knowledge and beliefs about HIV/AIDS that can inform decisions relating to the allocation of scarce resources. The tool will be very useful in the selection process of would-be educators particularly in health-promotion interventions.

Intracellular dynamics of HIV infection

Early studies of HIV infection dynamics suggested that virus-producing HIV-infected cells had an average half-life of approximately 1 day. However, whether this average behavior is reflective of the dynamics of individual infected cells is unclear. Here, we use HIV-enhanced green fluorescent protein (EGFP) constructs and flow cytometry sorting to explore the dynamics of cell infection, viral protein production, and cell death in vitro. By following the numbers of productively infected cells expressing EGFP over time, we show that infected cell death slows down over time. Although infected cell death in vivo could be very different, our results suggest that the constant decay of cell numbers observed in vivo during antiretroviral treatment could reflect a balance of cell death and delayed viral protein production. We observe no correlation between viral protein production and death rate of productively infected cells, showing that viral protein production is not likely to be the sole determinant of the death of HIV-infected cells. Finally, we show that all observed features can be reproduced by a simple model in which infected cells have broad distributions of productive life spans, times to start viral protein production, and viral protein production rates. This broad spectrum of the level and timing of viral protein production provides new insights into the behavior and characteristics of HIV-infected cells.