Development of real-time PCR for quantitation of simian immunodeficiency virus 2-LTR circles
Michael Lee1, Woong-Ki Kim2, Marcelo J Kuroda3
1Advanced BioScience Laboratories, Inc., Rockville, MD, USA.
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.
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