Koshizuka Tetsuo, Sato Yuko, Kobiyama Shoe, Oshima Mami, Suzutani Tatsuo
Department of Microbiology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Microbiol Immunol. 2018 Oct;62(10):651-658. doi: 10.1111/1348-0421.12645.
Quantification of human cytomegalovirus (HCMV) replication by plaque assay reflects viral infectivity but has several drawbacks. Recombinant virus expressing reporter genes can facilitate quantification of HCMV replication. In this study, a recombinant virus, Towne-BAC(dTT)-MetLuc, was constructed and the secretable Metridia luciferase (MetLuc) gene inserted into it under UL146 promoter. In addition, the UL130 gene was repaired to allow growth of the recombinant virus in both fibroblasts and epithelial cells. As predicted, luciferase activity was secreted into the culture medium and correlated with virus replication in infected fibroblasts and epithelial cells. Furthermore, secreted luciferase activity was correlated with the size of the recombinant virus inoculum with a dynamic range of 3 logs. This recombinant virus was used in a two-step culture protocol for detection of the anti-HCMV activity of compounds; that is, the supernatant of a first-step culture with anti-viral compounds was collected and inoculated into uninfected cells to create a second-step culture. Although secreted luciferase activity leaked in the first-step culture supernatant in the presence of some antiviral compounds, luciferase activity in the second-step culture supernatant reflected the virus yield in the first-step culture. Therefore, comparison of luciferase activity in the first- and second-step cultures indicated the anti-viral activity of the compounds. This two-step culture protocol facilitates screening of anti-viral compounds.
通过空斑试验对人巨细胞病毒（HCMV）复制进行定量分析反映了病毒的感染性，但存在若干缺点。表达报告基因的重组病毒有助于对HCMV复制进行定量分析。在本研究中，构建了一种重组病毒Towne-BAC(dTT)-MetLuc，并将可分泌的海洋发光酶（MetLuc）基因在UL146启动子的调控下插入其中。此外，对UL130基因进行了修复，以使重组病毒能够在成纤维细胞和上皮细胞中生长。正如预期的那样，发光酶活性分泌到培养基中，并与受感染的成纤维细胞和上皮细胞中的病毒复制相关。此外，分泌的发光酶活性与重组病毒接种物的大小相关，动态范围为3个对数级。这种重组病毒被用于一种两步培养方案，以检测化合物的抗HCMV活性；也就是说，收集含有抗病毒化合物的第一步培养的上清液，并接种到未感染的细胞中以建立第二步培养。尽管在某些抗病毒化合物存在的情况下，第一步培养上清液中会有分泌的发光酶活性泄漏，但第二步培养上清液中的发光酶活性反映了第一步培养中的病毒产量。因此，比较第一步和第二步培养中的发光酶活性可表明化合物的抗病毒活性。这种两步培养方案有助于筛选抗病毒化合物。
