Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil; Pós-graduação em Biologia Molecular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.
Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil.
J Virol Methods. 2018 Aug;258:49-53. doi: 10.1016/j.jviromet.2018.05.011. Epub 2018 May 22.
Human norovirus (HuNoV) is one of the main causes of acute gastroenteritis worldwide and is responsible for at least 20% of all cases. The detailed molecular mechanism of this norovirus remains unknown due to the lack of a suitable in vitro culturing system. An infectious clone of HuNoV would be a useful tool for elucidating the processes of viral infection and the mechanisms of replication. We developed an infectious cDNA clone of HuNoV using the rapid technique of Gibson Assembly. The complete genome of the HuNoV GII.4 Sydney subtype was cloned into a previously modified pcDNA3.1-based plasmid vector downstream from a cytomegaloviral promoter. We monitored the viral infection in vitro by inserting the reporter gene of the green fluorescent protein (GFP) between the NTPase and p22 genes, also by Gibson Assembly, to construct a HuNoV-GFP replicon. Human Caco-2 cells were transfected with the full-length genomic clone and the replicon containing GFP. The gene encoding the VP1/VP2 capsid protein was expressed, which was indirect evidence of the synthesis of subgenomic RNAs and thus the negative strand of the genome. We successfully constructed the infectious clone and its replicon containing GFP for the HuNoV GII.4 Sydney subtype, a valuable tool that will help the study of noroviral infection and replication.
人类诺如病毒（HuNoV）是全球急性胃肠炎的主要病因之一，至少占所有病例的 20%。由于缺乏合适的体外培养系统，这种诺如病毒的详细分子机制仍不清楚。HuNoV 的感染性克隆将是阐明病毒感染过程和复制机制的有用工具。我们使用 Gibson 组装的快速技术开发了 HuNoV 的感染性 cDNA 克隆。将 HuNoV GII.4 悉尼亚型的完整基因组克隆到先前修饰的基于 pcDNA3.1 的质粒载体中，位于巨细胞病毒启动子的下游。我们通过在 NTPase 和 p22 基因之间插入绿色荧光蛋白（GFP）的报告基因，也通过 Gibson 组装，构建了 HuNoV-GFP 复制子，从而在体外监测病毒感染。用全长基因组克隆和含有 GFP 的复制子转染人 Caco-2 细胞。表达了编码 VP1/VP2 衣壳蛋白的基因，这是亚基因组 RNA 合成的间接证据，因此也是基因组的负链。我们成功构建了 HuNoV GII.4 悉尼亚型的感染性克隆及其含有 GFP 的复制子，这是一种有价值的工具，将有助于研究诺如病毒的感染和复制。
