人类诺如病毒反向遗传体系的构建与验证

Construction and validation of human norovirus infectious clones

为验证已报道的能复制人类诺如病毒(human norovirus,HuNoV)GⅡ.3 U201感染性克隆的复制能力,进一步在HuNoV临床病毒株中寻找更优序列,构建能高效复制的感染性克隆,本研究合成含有T7启动子和EF-1α启动子的HuNoV GⅡ.3 U201全长序列,构建质粒pU201和pEF-1α-U201及对应的病毒RNA聚合酶失活的突变质粒。在COS 7细胞和Huh 7细胞中分别共转染T7聚合酶与pU201以及单独转染pEF-1α-U201,利用实时荧光定量聚合酶链反应检测转染后不同时间点细胞内病毒RNA水平。结果显示,与对照组相比,pU201和pEF-1α-U201的病毒RNA水平升高约2倍和3倍。为便于检测HuNoV复制,在pU201和pEF-1α-U201质粒中分别插入NanoLuc TM荧光素酶报告基因,并命名为pU201-Nluc和pEF-1α-U201-Nluc。将质粒分别转染COS 7细胞和Huh 7细胞,检测转染后不同时间点Nluc荧光素酶活性。结果显示,pEF-1α-U201-Nluc的Nluc荧光素酶活性相较于对照组增高近2倍,而pU201-Nluc无明显升高,提示EF-1α启动子起始病毒复制优于T7启动子。为寻找优于HuNoV GⅡ.3 U201的HuNoV序列,合成HuNoV GⅡ.4临床病毒株全长序列,将其插入EF-1α启动子载体中并转染细胞,于不同时间点检测上清及细胞中病毒RNA水平。结果显示,上清及细胞中病毒RNA水平与对照组相比无显著差异。以上结果提示,已报道可复制的HuNoV GⅡ.3 U201反向遗传体系复制效率有限,且HuNoV反向遗传体系复制能力可能与不同序列的病毒相关。

The lack of a cultivation system for human noroviruses(HuNoV)is a major barrier to understand virus biology and the development of effective antiviral strategies.The study aims to verify the reported replication of the reverse genetics system of HuNoV GⅡ.3 U201,and further to seek a better sequence from the HuNoV clinical strains to construct a highly efficient infectious clone.First the sequences of HuNoV GⅡ.3 U201 genome with T7 promoter and EF-1αpromoter were synthesized,and inserted into the vector,named pU201 and pEF-1α-U201,respectively.Meanwhile,negative control plasmids with viral RNA polymerase activity inactivated were constructed.To verify whether these clones could replicate,T7 polymerase was co-transfected with pU201 and pEF-1α-U201 was transfected alone in COS 7 cells and Huh 7 cells,respectively,and HuNoV RNA level was determined by real-time quantitative polymerase chain reaction(RT-qPCR)in different time post transfection.The results showed that the HuNoV RNA levels of pU201 and pEF-1α-U201 were nearly two times and three times higher than those of the negative control group respectively.To facilitate the detection of HuNoV replication,full-length infectious clones with NanoLuc TM luciferase(Nluc)reporter gene were constructed by inserting Nluc sequence in front of viral genome,named pU201-Nluc and pEF-1α-U201-Nluc.COS 7 cells and Huh 7 cells were transfected and Nluc activity was determined in different time post transfection.The results showed that the Nluc activity of pEF-1α-U201-Nluc was nearly 2 times higher than those of the negative control group and the inhibitor-treated group,but there was no significant increase in pU201-Nluc.These results suggested that EF-1αpromoter was superior to T7 promoter in initiating HuNoV GⅡ.3 U201 replication.Finally,to seek a better HuNoV sequence,a full-length infectious clone was constructed with a clinical isolate of HuNoV GⅡ.4 genome from a Taiwan patient and EF-1αpromoter,named PCTc.COS 7 cells and Huh 7 cells were transfected and HuNoV RNA level was determined in supernatant and cells by RT-qPCR.And the viral RNA level between PCTc and control groups had no significant difference.The results suggested that full-length infectious clones and subgenomic clone of HuNoV GⅡ.3 U201 could only achieve limited and unsustainable replication in cells,and replication capacity may also be associated with different sequences of virus strains.