Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons

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Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons

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dc.contributor.author Bruce, A Gregory en_US
dc.contributor.author Bakke, Angela M. en_US
dc.contributor.author Thouless, Margaret E. en_US
dc.contributor.author Rose, Timothy M. en_US
dc.date.accessioned 2010-05-06T20:05:10Z
dc.date.available 2010-05-06T20:05:10Z
dc.date.issued 2005 en_US
dc.identifier.citation Bruce AG, Bakke A, Thouless M, Rose T. Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons. Virology Journal. 2005;2(1):2. en_US
dc.identifier.other 10.1186/1743-422X-2-2 en_US
dc.identifier.uri http://www.virologyj.com/content/2/1/2 en_US
dc.identifier.uri http://hdl.handle.net/1773/15836
dc.description.abstract Background: Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) have been identified in macaques and other Old World non-human primates. We have developed a real-time quantitative PCR (QPCR) assay using a TaqMan probe to differentially detect and quantitate members of the rhadinovirus-2 (RV2) lineage. PCR primers were derived from sequences within ORF 60 and the adjacent ORF 59/60 intergenic region which were highly conserved between the macaque RV2 rhadinoviruses, rhesus rhadinovirus (RRV) and Macaca nemestrina rhadinovirus-2 (MneRV2). These primers showed little similarity to the corresponding sequences of the macaque RV1 rhadinoviruses, retroperitoneal fibromatosis herpesvirus Macaca nemestrina (RFHVMn) and Macaca mulatta (RFHVMm). To determine viral loads per cell, an additional TaqMan QPCR assay was developed to detect the single copy cellular oncostatin M gene. Results: We show that the RV2 QPCR assay is linear from less than 2 to more than 300,000 copies using MneRV2 DNA, and is non-reactive with RFHVMn DNA up to 1 billion DNA templates per reaction. RV2 loads ranging from 6 to 2,300 viral genome equivalent copies per 106 cells were detected in PBMC from randomly sampled macaques from the Washington National Primate Research Center. Screening tissue from other primate species, including another macaque, Macaca fascicularis, and a baboon, Papio cynocephalus, revealed the presence of novel rhadinoviruses, MfaRV2 and PcyRV2, respectively. Sequence comparison and phylogenetic analysis confirmed their inclusion within the RV2 lineage of KSHV-like rhadinoviruses. Conclusions: We describe a QPCR assay which provides a quick and sensitive method for quantitating rhadinoviruses belonging to the RV2 lineage of KSHV-like rhadinoviruses found in a variety of macaque species commonly used for biomedical research. While this assay broadly detects different RV2 rhadinovirus species, it is unreactive with RV1 rhadinovirus species which commonly co-infect the same primate hosts. We also show that this QPCR assay can be used to identify novel RV2 rhadinoviruses in different primate species. en_US
dc.description.sponsorship This work was partially supported by RR13154 and RR00166 from the National Center for Research Resources. T. Rose is the recipient of a K02 award, AI49275, from the National Institute for Allergy and Infectious Diseases. en_US
dc.language.iso en_US en_US
dc.title Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons en_US
dc.type Article en_US


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