A two-step culture method utilizing secreted luciferase recombinant virus for detection of anti-cytomegalovirus compounds
A two-step culture method utilizing secreted luciferase recombinant virus for detection of anti-cytomegalovirus compounds
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利用分泌型荧光素酶重组病毒的两步培养法检测抗巨细胞病毒化合物
DOI：
10.1111/1348-0421.12645
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发表时间：
2018
影响因子：
2.6
通讯作者：
Suzutani Tatsuo
中科院分区：
文献类型：
--
作者：
Koshizuka Tetsuo;Sato Yuko;Kobiyama Shoe;Oshima Mami;Suzutani Tatsuo
通过空斑测定来定量人巨细胞病毒（HCMV）复制反映了病毒感染性，但有几个缺点。表达报告基因的重组病毒可促进HCMV复制的定量。本研究构建了重组病毒Towne-BAC（dTT）-MetLuc，并将分泌型Metridialuciferase（MetLuc）基因插入到重组病毒UL 146启动子下。此外，修复了UL 130基因，使重组病毒能够在成纤维细胞和上皮细胞中生长。正如预测的那样，荧光素酶活性分泌到培养基中，并与感染的成纤维细胞和上皮细胞中的病毒复制相关。此外，分泌的荧光素酶活性与重组病毒接种物的大小相关，动态范围为3 log。该重组病毒用于两步培养方案中，用于检测化合物的抗HCMV活性;即，收集含有抗病毒化合物的第一步培养物的上清液，并接种到未感染的细胞中，以产生第二步培养物。尽管在存在一些抗病毒化合物的情况下，第一步培养上清中分泌的荧光素酶活性泄漏，但第二步培养上清中的荧光素酶活性反映了第一步培养中的病毒产量。因此，第一步和第二步培养物中荧光素酶活性的比较表明化合物具有抗病毒活性。这种两步培养方案有助于筛选抗病毒化合物。
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 secretableMetridialuciferase (MetLuc) gene inserted into it underUL146promoter. In addition, theUL130gene 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.