Dengue virus (DENV) is the causative agent of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) and continues to be a public health problem in the tropical and subtropical areas. However, there is currently no antiviral treatment for DENV infection. In this study, our aim was to develop a stable reporter replicon cell system that supports constant viral RNA replication in cultured cells. The isolated replicon cells exhibited high levels of luciferase activity in the culture supernatant concomitant with expression of virus-encoded NS1, NS3 and NS5 proteins in the cells. The NS1, NS3 proteins and dsRNA were detected in the replicon cells by immunofluorescence analysis. Furthermore, the anti-DENV inhibitors ribavirin and bromocriptine significantly reduced the luciferase activity in a dose-dependent manner. High-throughput screening with a compound library using the stably-transfected replicon cells showed a Z' factor value of 0.57. Our screening yielded several candidates including one compound that has already shown anti-DENV activity. Taken together, our results demonstrate that this DENV subgenomic replicon cell system expressing a secretory luciferase gene can be useful for the high-throughput screening of anti-DENV compounds and the analysis of the replication mechanism of the DENV RNA.

中文翻译：

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使用表达分泌型荧光素酶的复制子细胞鉴定登革热病毒复制的抑制剂。

登革热病毒（DENV）是登革热（DF），登革出血热（DHF）和登革热休克综合征（DSS）的病原体，并继续是热带和亚热带地区的公共卫生问题。但是，目前没有针对DENV感染的抗病毒治疗。在这项研究中，我们的目的是开发一种稳定的报告子复制子细胞系统，以支持在培养细胞中不断进行病毒RNA复制。分离的复制子细胞在培养上清液中表现出高水平的萤光素酶活性，并伴随着细胞中病毒编码的NS1，NS3和NS5蛋白的表达。通过免疫荧光分析在复制子细胞中检测到NS1，NS3蛋白和dsRNA。此外，抗DENV抑制剂利巴韦林和溴隐亭以剂量依赖性方式显着降低了萤光素酶活性。使用稳定转染的复制子细胞通过化合物库进行的高通量筛选显示Z'因子值为0.57。我们的筛选产生了几种候选物，包括一种已经显示出抗DENV活性的化合物。两者合计，我们的结果表明，表达分泌型荧光素酶基因的DENV亚基因组复制子细胞系统可用于高通量筛选抗DENV化合物和分析DENV RNA的复制机制。