Development of a generic real-time PCR assay for simultaneous detection of proviral DNA of simian Betaretrovirus serotypes 1, 2, 3, 4 and 5 and secondary uniplex assays for specific serotype identification

Abstract

Simian betaretroviruses, (formerly Type D retroviruses; SRV) are a group of closely related retroviruses for which the natural host species are Asian monkeys of the genus Macaca. Five serotypes have been identified by classical neutralization assays and three additional untyped variants have been reported (SRV Tsukuba , SRV-6, SRV-7). These viruses may be significant pathogens in macaque colonies, causing a broad spectrum of clinical disease secondary to viral-induced immune suppression. Undetected SRV infections in research macaques also represent a potential confounding variable in research protocols and a concern for human caretakers. Intensive testing efforts have been implemented to identify infected animals in established colonies. A real-time quantitative generic multiplex PCR assay was developed that is capable of simultaneous detection of proviral DNA of SRV serotypes 1, 2, 3, 4 and 5. This assay incorporates amplification of the oncostatin M (OSM) gene for confirmation of amplifiable DNA and allows quantitation of the number of proviral copies per cell analyzed in each multiplex reaction. Detection of multiple serotypes by PCR increases the efficiency and cost-effectiveness of SRV screening programs. A panel of SRV serotype-specific uniplex real-time PCR assays for discrimination among the 5 recognized serotypes is also described.

Cellular Immune Responses against Simian T-Lymphotropic Virus Type 1 Target Tax in Infected Baboons

“…PCR amplification of STLV-1 sequences was carried out by the California National Primate Research Center Pathogen Detection Laboratory. The procedure was based on a real-time quantitative PCR assay developed for simian betaretrovirus proviral DNA detection (49). In brief, PBMCs were separated by Ficoll density gradient centrifugation and frozen.…”

Hemophagocytic Syndrome in a Pancytopenic Simian Retrovirus–Infected Male Rhesus Macaque (Macaca mulatta)

“…Real-time PCR for SRV was performed on spleen (frozen) and bone marrow (formalin fixed) using primers and probe targeting the env gene. 25 The animal was positive by PCR for SRV in both tissues. Further PCR established the serotype as SRV type 2.…”

ComprehensiveIn VitroAnalysis of Simian Retrovirus Type 4 Susceptibility to Antiretroviral Agents

“…The viral copy number was quantified by using the One Step PrimeScript reverse transcriptase PCR (RT-PCR) kit (TaKaRa, Otsu, Japan) and the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) with a known copy number control. The primer sets and a probe used for SRV-4 amplification were described previously (20). PCR conditions were 5 min at 42°C, 10 s at 95°C, and 55 cycles of 5 s at 95°C and 34 s at 62°C.…”