A genetic system for rhesus monkey rhadinovirus: Use of recombinant virus to quantitate antibody-mediated neutralization
A genetic system for rhesus monkey rhadinovirus: Use of recombinant virus to quantitate antibody-mediated neutralization
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DOI：
10.1128/jvi.80.3.1549-1562.2006
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发表时间：
2006-02-01
影响因子：
5.4
通讯作者：
Desrosiers, RC
中科院分区：
文献类型：
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作者：
Bilello, JP;Morgan, JS;Desrosiers, RC
恒河猴流感病毒(RRV)是一种与Kaposi肉瘤相关疱疹病毒关系密切的猿猴伽玛-2疱疹病毒，它在培养的恒河猴成纤维细胞中裂解复制，并在B细胞中建立持久性。产生了重叠的粘粒克隆，覆盖了RRV 26-95株整个130千碱基对的基因组，包括其复制所需的末端重复区域。克隆的RRV是通过与跨越整个RRV26-95基因组的重叠宇宙共转染而产生的，复制的生长动力学和滴度与亲本的、未克隆的野生型RRV26-95相似。将分泌型工程碱性磷酸酶(SEAP)和绿色荧光蛋白(GFP)表达盒插入到RRV R1基因的上游，利用粘粒重组系统构建可复制的重组RRV，表达SENP或GFP报告基因。利用SEAP和GFP重组RRV，建立了检测RRV感染、中和和复制的方法。用恒河猴成纤维细胞检测自然或实验感染RRV的恒河猴血清中和RRV-SEAP和RRV-GFP感染性的能力。RRV阳性猴子的血清，而不是RRV阴性猴子的血清，在通过产生SEAP活性或表达GFP的能力进行检测时，始终能够中和RRV的传染性。中和活性存在于免疫球蛋白部分。在测试的17只恒河猴中，来自恒河猴26-95的血清，即产生RRV 26-95分离株的猴子，对RRV26-95具有最高效价的中和活性。这种基于粘粒的遗传系统和报告病毒中和试验将有助于研究单个RRV糖蛋白对进入不同类型的细胞，特别是成纤维细胞和B细胞的贡献。
Rhesus monkey rhadinovirus (RRV), a simian gamma-2 herpesvirus closely related to the Kaposi sarcoma-associated herpesvirus, replicates lytically in cultured rhesus monkey fibroblasts and establishes persistence in B cells. Overlapping cosmid clones were generated that encompass the entire 130-kilobase-pair genome of RRV strain 26-95, including the terminal repeat regions required for its replication. Cloned RRV that was produced by cotransfection of overlapping cosmids spanning the entire RRV26-95 genome replicated with growth kinetics and to titers similar to those of the parental, uncloned, wild-type RRV26-95. Expression cassettes for secreted-engineered alkaline phosphatase (SEAP) and green fluorescent protein (GFP) were inserted upstream of the R1 gene, and the cosmid-based system for RRV genome reconstitution was used to generate replication-competent, recombinant RRV that expressed either the SENP or GFP reporter gene. Using the SEAP and GFP recombinant RRVs, assays were developed to monitor RRV infection, neutralization, and replication. Heat-inactivated sera from rhesus monkeys that were naturally or experimentally infected with RRV were assayed for their ability to neutralize RRV-SEAP and RRV-GFP infectivity using rhesus monkey fibroblasts. Sera from RRV-positive monkeys, but not RRV-negative monkeys, were consistently able to neutralize RRV infectivity when assayed by the production of SEAP activity or by the ability to express GFP. The neutralizing activity was present in the immunoglobulin fraction. Of the 17 rhesus monkeys tested, sera from rhesus monkey 26-95, i.e., the monkey that yielded the RRV 26-95 isolate, had the highest titer of neutralizing activity against RRV26-95. This cosmid-based genetic system and the reporter virus neutralization assay will facilitate study of the contribution of individual RRV glycoproteins to entry into different cell types, particularly fibroblasts and B cells.