Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons
Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons
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DOI：
10.1186/1743-422x-2-2
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发表时间：
2005-01-01
期刊：
影响因子：
4.8
通讯作者：
Rose, Timothy M.
中科院分区：
文献类型：
--
作者：
Bruce, A. Gregory;Bakke, Angela M.;Rose, Timothy M.
背景：已在猕猴和其他旧大陆非人类灵长类动物中鉴定出与卡波西肉瘤相关疱疹病毒 (KSHV/HHV8) 相关的两种不同的鼻病毒谱系。我们开发了一种实时定量 PCR (QPCR) 检测方法，使用 TaqMan 探针来差异检测和定量 rhadinovirus-2 (RV2) 谱系的成员。 PCR 引物源自 ORF 60 和相邻 ORF 59/60 基因间区域内的序列，这些序列在猕猴 RV2 病毒、恒河猴病毒 (RRV) 和猕猴 线虫病毒-2 (MneRV2) 之间高度保守。这些引物与猕猴 RV1 鼻病毒、腹膜后纤维瘤疱疹病毒猕猴 (RFHVMn) 和猕猴 (RFHVMm) 的相应序列几乎没有相似性。为了确定每个细胞的病毒载量，开发了一种额外的 TaqMan QPCR 测定法来检测单拷贝细胞制瘤素 M 基因。结果：我们表明，使用 MneRV2 DNA，RV2 QPCR 测定法在少于 2 至超过 300,000 个拷贝之间呈线性，并且每次反应与 RFHVMn DNA 不发生反应，最多 10 亿个 DNA 模板。从华盛顿国家灵长类研究中心随机抽取的猕猴的 PBMC 中检测到每 10(6) 个细胞 RV2 载量范围为 6 至 2,300 个病毒基因组等效拷贝。对其他灵长类动物（包括另一种猕猴（Macaca fasciculis）和狒狒（Papio cynocephalus）的组织进行筛选，结果分别揭示了新型鼻病毒 MfaRV2 和 PcyRV2 的存在。序列比较和系统发育分析证实它们包含在 KSHV 样鼻病毒的 RV2 谱系中。结论：我们描述了一种 QPCR 检测方法，该方法提供了一种快速、灵敏的方法来定量属于生物医学研究常用的多种猕猴物种中发现的 KSHV 样鼻病毒 RV2 谱系的鼻病毒。虽然该检测广泛检测不同的 RV2 病毒种类，但它与通常共同感染相同灵长类宿主的 RV1 病毒种类不发生反应。我们还表明，这种 QPCR 检测可用于鉴定不同灵长类动物中的新型 RV2 病毒。
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 10(6) 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.