Objectives In order to gain insight into the determinants of nonpathogenic SIV infection in the naturally infected sooty mangabey host, SIV replication in mangabeys is assessed and compared to SIV replication observed in pathogenically infected rhesus macaques. Concurrent with Dr. Staprans move from the University of California to Emory University, significant methodologic progress was made in two areas 1) Implementation of a kinetic PCR method for the high throughput quantitation of SIV nucleic acid in infected monkeys. Real-time PCR-based assays for SIV RNA were implemented. The assays have the capability to detect a variety of SIV isolates, including SIVmac-related and SIVsmm viruses. Two different primer/probe sets have been implemented and evaluated with respect to assay sensitivity, linearity, reproducibility, and ability to detect divergent viral variants. Well-characterized and independently quantified SIV virion standards for an external standard curve ha ve also been implemented. The standards were used to identify optimal RT-PCR conditions. An assay based on 5'LTR sequences has a sensitivity of 100 copies SIV RNA/ml plasma with a 0.15 ml plasma input, a linear dynamic range of 100 to 108 copies SIV RNA (or DNA), and an average coefficient of variation <20%. Our ability to rapidly develop quantitative, real-time PCR assays for both viral DNA and RNA demonstrates that this methodology can be readily applied to the analysis of virtually any RNA or DNA virus. Application of radiolabelled SIV in situ hybridization probes demonstrated the increased sensitivity of this method compared to non-isotopic methods previously used. The enhanced sensitivity has demonstrated similar or slightly lower frequency of SIV-infected T cells in mangabey lymphoid tissues as compared to macaques. Confirmatory studies to extend these observations are in progress. FUNDING NIH / NIAID $131,526 5/01/96 - 4/30/01 PUBLICATIONS None P51RR00165-38 1/1/1998 - 12/31/1998 Yerkes Regional Primate Research Center
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