Construction and validation of human norovirus infectious clones
CHEN Shuiye1, SONG Wuhui1，YI Zhigang1，2
1. Department of Medical Microbiology and Parasitology, Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; 2. Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
Abstract：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 G Ⅱ. 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 EF1α promoter were synthesized, and inserted into the vector, named pU201 and pEF1αU201, respectively. Meanwhile, negative control plasmids with viral RNA polymerase activity inactivated were constructed. To verify whether these clones could replicate, T7 polymerase was cotransfected with pU201 and pEF1αU201 was transfected alone in COS 7 cells and Huh 7 cells, respectively, and HuNoV RNA level was determined by realtime quantitative polymerase chain reaction (RTqPCR) in different time post transfection. The results showed that the HuNoV RNA levels of pU201 and pEF1αU201 were nearly two times and three times higher than those of the negative control group respectively. To facilitate the detection of HuNoV replication, fulllength infectious clones with NanoLuc™ luciferase (Nluc) reporter gene were constructed by inserting Nluc sequence in front of viral genome, named pU201Nluc and pEF1αU201Nluc. 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 pEF1αU201Nluc was nearly 2 times higher than those of the negative control group and the inhibitortreated group, but there was no significant increase in pU201Nluc. These results suggested that EF1α promoter was superior to T7 promoter in initiating HuNoV GⅡ.3 U201 replication. Finally, to seek a better HuNoV sequence, a fulllength infectious clone was constructed with a clinical isolate of HuNoV GⅡ.4 genome from a Taiwan patient and EF1α promoter, named PCTc. COS 7 cells and Huh 7 cells were transfected and HuNoV RNA level was determined in supernatant and cells by RTqPCR. And the viral RNA level between PCTc and control groups had no significant difference. The results suggested that fulllength 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.
陈水叶1，宋武慧1，易志刚1，2. 人类诺如病毒反向遗传体系的构建与验证[J]. 微生物与感染, 2021, 16(2): 71-78.
CHEN Shuiye1, SONG Wuhui1，YI Zhigang1，2. Construction and validation of human norovirus infectious clones. JOURNAL OF MICROBES AND INFECTIONS, 2021, 16(2): 71-78.