Development of real-time PCR assays for quantitation of simian betaretrovirus serotype-1, -2, -3, and -5 viral DNA in Asian monkeys.

Simian betaretroviruses (SRV), formerly known as simian type D retroviruses, are endemic in many populations of Asian monkeys of the genus Macaca. Asian monkeys have been used extensively as animal models for preclinical HIV vaccine development, therapeutics, and other biomedical studies. SRV infection can sometimes lead to immune deficiency disease, which complicates such studies; thus, it is important to screen for SRV infection and remove infected animals from test populations. Real-time PCR assays were developed to specifically quantify SRV-1/3, SRV-2, and SRV-5 proviral DNA. The SRV provirus copy numbers were standardized relative to real-time PCR measurements of the rhesus macaque albumin gene. The primers and TaqMan probe sequences for the rhesus macaque (Indian origin) albumin gene also detect cynomolgus macaque and rhesus macaque (Chinese origin) albumin genes. The SRV primers and probes were designed to amplify gag gene sequences of SRV-1/3 (GeneBank accession number M11841), SRV-2 (GeneBank accession number M16605), and SRV-5 (GeneBank accession number AF252389). The optimized reactions for detection of each SRV serotype and the macaque albumin gene had amplification efficiencies of greater than 90% with a linear range spanning 1×101 to 2.5×106 copies per reaction. The R2 values of all standard curves were greater than 0.995. Of 40 animals housed in quarantine, four animals were positive for SRV-1/3 with 28, 5450, 9780, and 14,500 copies of provirus per 106 PBMCs, and one animal was positive for SRV-2 with provirus copy number of 7790 per 106 PBMCs. All of 40 animals appeared to be seronegative and had normal CD4+ and CD8+ T-cell counts. These quantitative real-time PCR assays enhance the detection and quantitation of SRV infection and will facilitate the elimination of this virus from macaque colonies.