Researchers have developed a new sophisticated sensor that detects HIV hiding places inside and outside the cells. "It is a fantastic new technology that will allow us to visualize the virus in tissues that we have 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 of this molecular microscope high power, revealed at an international AIDS conference last week, may clarify critical questions about the persistence of HIV and ultimately on how to rid the body of the virus.
To date, HIV assessments in the fabric known as in situ-analysis was hampered by a major difficulty. The most common sensors, which use fluorescent labels or radioactive markers to pinpoint the location of the virus in a tissue sample, sometimes have difficulty distinguishing the target RNA and HIV DNA from surrounding cell components . In essence, a marker can mislabel tissue the virus, creating a background that throws the analysis. The new technique has "very little noise," says immunologist Jake Estes National Frederick of the National Cancer Institute Laboratory (sister of NIAID) in Frederick, Maryland, used to produce highly detailed images of the AIDS virus in various tissues monkey (above) submitted to the conference.
Estes developed the technique in collaboration with Advanced Cell Diagnostics Hayward, California, modifying the existing product RNAscope of the company to detect HIV RNA, DNA or both the same time. RNA and DNA are composed of nucleotides that complement pair with a guanine, for example, binds to cytosine. Traditional methods for long strings of HIV use mapping the genetic material of these nucleotides, called oligomers, to find and bind to complementary strands of DNA or RNA in the sample tissue. These oligomers are labeled with a marker so they send a signal when they have reached their target, allowing the researchers to create an accurate picture of where the viral genetic material is dispersed in the tissue sample. But the oligomers are molecules large and somewhat clumsy, and they sometimes bind to cellular components other than the target sequence.
new technique Estes, however, uses a more complex sensor system that all but eliminates such errors. In essence, the approach chops in a two oligomer and sends the two halves in search of the target sequence. Their markers on if an additional oligomer which connects the two halves binds to time, which occurs only when they are parked next to each other on the target. The probability is extremely low that the two probes would land next to each other on something other than HIV.
HIV is an RNA virus, but it also converts to a form of DNA that allows it to build its genes in a human chromosome. Estes, who works with the virologist Jeffrey Lifson, also developed a DNAscope to view this DNA HIV-called provirus, which becomes embedded in human cells and can persist for decades without being attacked by the immune or antiretroviral system (ARV) . "Reservoirs" of infected cells that hold latent proviral are a major reason powerful combinations of ARVs can not eliminate infection and heal people.
Estes, Lifson and colleagues monkeys infected with the simian version of the AIDS virus and analyzed the tissues from many parts of their bodies. And RNAscope DNAscope were able to distinguish cells that harbor the provirus, the viral RNA, or even viruses outside cells much more clearly than any in situ prior art. "We are convinced that we can see individual virions, and it has an exquisite sensitivity and specificity," said Estes. To check their work, they counted HIV virions by the eye in one of their new images, and then compared the total to a validated measure of viral levels. "We see a good correlation," said Estes.
HIV / AIDS researchers working to heal the face of infection several obstacles that these new fields could help to overcome. The first is the absence of detectable virus in the blood plasma of patients on effective ART, making it difficult for researchers to assess whether an intervention to cure the infection works. Several techniques exist to measure changes in the tanks, but each has shortcomings that new tracts would be able to complete. Another obstacle is to not know where in the body the provirus prefers to hide. If the new sensor can help solve this longstanding puzzle, they could refine the retraction attempts viral reservoirs. "If we can go and see what happens to the virus in these tissues with this kind of sensitivity and specificity, it will answer a lot of questions," says NIAID Koup.
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