Bilello John P, Morgan Jennifer S, Damania Blossom, Lang Sabine M, Desrosiers Ronald C
New England Primate Research Center/Harvard Medical School, One Pine Hill Drive, P.O. Box 9102, Southborough, MA 01772-9102, USA.
J Virol. 2006 Feb;80(3):1549-62. doi: 10.1128/JVI.80.3.1549-1562.2006.
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 SEAP 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.
恒河猴疱疹病毒（RRV）是一种与卡波西肉瘤相关疱疹病毒密切相关的猿γ-2疱疹病毒，它在培养的恒河猴成纤维细胞中进行裂解复制，并在B细胞中建立持续性感染。构建了重叠黏粒克隆，其包含RRV 26-95株完整的130千碱基对基因组，包括其复制所需的末端重复区域。通过共转染跨越整个RRV26-95基因组的重叠黏粒产生的克隆RRV，其生长动力学和滴度与亲本、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）的表达盒插入R1基因上游，并使用基于黏粒的RRV基因组重建系统来产生表达SEAP或GFP报告基因的具有复制能力的重组RRV。利用SEAP和GFP重组RRV，开发了用于监测RRV感染、中和及复制的检测方法。使用恒河猴成纤维细胞，检测了自然感染或实验感染RRV的恒河猴的热灭活血清中和RRV-SEAP和RRV-GFP感染性的能力。当通过SEAP活性的产生或表达GFP 的能力进行检测时，来自RRV阳性猴而非RRV阴性猴的血清始终能够中和RRV感染性。中和活性存在于免疫球蛋白组分中。在测试的17只恒河猴中，来自恒河猴26-95（即产生RRV 26-95分离株的猴子）的血清对RRV26-95具有最高滴度的中和活性。这种基于黏粒的遗传系统和报告病毒中和检测将有助于研究单个RRV糖蛋白在进入不同细胞类型（特别是成纤维细胞和B细胞）中的作用。
