Researchers have developed a sophisticated new probe that detects HIV’s hiding places inside and outside of cells. “It’s a fantastic new technique that’s going to allow us to visualize the virus in tissues like we’ve never been able to before,” says immunologist Richard Koup, deputy director of the Vaccine Research Center at the National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland, who was not involved in the research. Insights from this high-powered molecular microscope, revealed at an international AIDS conference last week, may clarify critical questions about HIV persistence and, ultimately, about how to rid the body of the virus.

To date, assessments of HIV in tissue—known as in situ analysis—have been hampered by one major difficulty. The most common probes, which use fluorescent markers or radioactive labels to pinpoint the virus’s location in a tissue sample, sometimes have difficulty distinguishing the target—HIV RNA and DNA—from surrounding cellular components. In essence, a marker can mislabel cell tissue as the virus, creating background noise that throws off the analysis. The new technique has “very little noise,” says immunologist Jake Estes of the Frederick National Laboratory of the National Cancer Institute (a sister of NIAID) in Frederick, Maryland, who used it to produce highly detailed images of the AIDS virus in various monkey tissues (above) that he presented at the conference.

Estes developed the technique in collaboration with Advanced Cell Diagnostics of Hayward, California, by modifying the company’s already existing RNAscope product to detect HIV RNA, DNA or both at the same time. RNA and DNA are made of nucleotides that pair with a complement—guanine, for example, binds to cytosine. Traditional methods for mapping HIV genetic material use long strings of these nucleotides, called oligomers, to find and bind to complementary strands of DNA or RNA in sample tissues. These oligomers are labeled with a marker so they send a signal when they hit their target, allowing researchers to create an image of precisely where the viral genetic material is dispersed throughout the tissue sample. But oligomers are large and somewhat clumsy molecules, and they occasionally bind to cellular components other than the target sequence.

Estes’s new technique, in contrast, uses a more complex probe system that all but eliminates those kinds of errors. In essence, the approach chops an oligomer in two and sends both halves out to find the target sequence. Their markers light up if an additional oligomer that bridges the two halves binds to both, which only occurs when they park next door to each other on the target. The probability is extremely low that the two probes would land next to each other on anything other than HIV.

HIV is an RNA virus, but it also converts to a DNA form that allows it to weave its genes into a human chromosome. Estes, who works with virologist Jeffrey Lifson, has also developed a DNAscope to visualize this HIV DNA—called the provirus—which becomes integrated into human cells and can persist for decades without being attacked by the immune system or antiretroviral (ARV) drugs. “Reservoirs” of infected cells that hold latent provirus are a key reason why powerful combinations of ARVs cannot eliminate infections and cure people.

Estes, Lifson, and co-workers infected monkeys with the simian version of the AIDS virus and then analyzed tissues from many parts of their bodies. Their RNAscope and DNAscope were able to distinguish cells that harbor the provirus, viral RNA, or even viruses outside of cells much more clearly than any previous in situ technique. “We’re convinced that we can see individual virions and that this has exquisite sensitivity and specificity,” Estes says. To double check their work, they counted HIV virions by eye in one of their new images, and then compared their count to a validated measure of viral levels. “We see a beautiful correlation,” Estes says.

HIV/AIDS researchers working to cure the infection face several obstacles that these new scopes could help overcome. One is the lack of detectable virus in the blood plasma of patients on effective ARV therapy, which makes it difficult for researchers to assess whether an intervention aimed at curing the infection is working. Several techniques exist to measure changes in reservoirs, but each has shortcomings that the new scopes might be able to supplement. Another obstacle is not knowing precisely where in the body the provirus prefers to hide. If the new probes can help solve this longstanding riddle, they could refine attempts to shrink viral reservoirs. “If we can go in and see what happens to the virus in these different tissues with this sort of sensitivity and specificity, it’s going to answer a lot of questions,” NIAID’s Koup says.