Development of real-time PCR for quantitation of simian immunodeficiency virus 2-LTR circles.

BACKGROUND Non-human primates infected with simian immunodeficiency virus (SIV) represent a robust model to evaluate pre-clinical efficacy of HIV-1 preventive strategies and to determine the size of reservoir. METHODS We developed a real-time qPCR assay to specifically quantify episomal 2-LTR circular DNA in peripheral blood mononuclear cells and brain tissues from SIV-infected macaques. RESULTS This assay has sensitivity, accuracy and reproducibility over seven orders of magnitude. High copy numbers of SIV 2-LTR circles were correlated to high proviral DNA levels in brains of two SIV encephalitic animals. In contrast, no 2-LTR circles were detectable in two SIV-infected animals with no sign of encephalitis or two animals that had mild encephalitis with low levels of proviral DNA. CONCLUSIONS These results suggest that simultaneous application of total proviral DNA and 2-LTR circle assays provides quantitative evaluation of pathogenesis and outcome of SIV infection in macaques.