|
|
|
| Construction and validation of human norovirus infectious clones |
| Shui-Ye CHEN1,Zhigang Yiwuhui SONG3 |
|
|
|
|
Abstract Abstract: The lack of a cultivation system for human noroviruses (HuNoVs) was 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 HuNoVs G Ⅱ. 3 U201, and further to seek a better sequence from the HuNoVs clinical strains to construct a highly efficient infectious clone. First the sequences of HuNoVs GⅡ.3 U201 genome with T7 promoter and EF-1α promoter were synthesized, and inserted into the vector respectively, named pU201 and pEF-1α-U201. 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 HuNoVs RNA level were determined by RT-qPCR in different time post transfection. The results shown that the HuNoV RNA levels of pU201 and pEF-1α-U201 were nearly 2 times higher than those of the negative control group respectively. To facilitate the detection of HuNoVs replication, full-length infectious clones with NanoLuc? luciferase (Nluc) reporter gene was 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 shown 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-1α promoter was superior to T7 promoter in initiating HuNoVs GⅡ.3 U201 replication. Finally, to seek a better HuNoVs’ sequence, a full-length infectious clone was constructed with a clinical isolate of HuNoV GⅡ.4 genome from a Taiwan patient and EF -1α promoter, 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 HuNoVs 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.
|
|
Received: 04 January 2021
Published: 01 January 2021
|
|
|
|
Corresponding Authors:
Zhigang Yi
|
|
|
|
| [1] |
DUIZER E, SCHWAB K J, NEILL F H, et al.Laboratory efforts to cultivate noroviruses[J].J Gen Virol, 2004, 85(Pt 1):79-87
|
| [2] |
VINJE J.Advances in laboratory methods for detection and typing of norovirus[J].J Clin Microbiol, 2015, 53(2):373-81
|
| [3] |
GUIX S, ASANAKA M, KATAYAMA K, et al.Norwalk virus RNA is infectious in mammalian cells[J].J Virol, 2007, 81(22):12238-48
|
| [4] |
GREEN K Y.Norovirus infection in immunocompromised hosts[J].Clin Microbiol Infec, 2014, 20(8):717-23
|
| [5] |
TRIVEDI T K, DESAI R, HALL A J, et al.Clinical characteristics of norovirus-associated deaths: A systematic literature review[J].Am J Infect Control, 2013, 41(7):654-7
|
| [6] |
YU J M, LIANG Z Y, GUO K, et al.Intra-Host Evolution of Norovirus GII.4 in a Chronic Infected Patient With Hematopoietic Stem Cell Transplantation[J].Front Microbiol, 2020, 11(375):1-9
|
| [7] |
DE GRAAF M, VAN BEEK J, KOOPMANS M P.Human norovirus transmission and evolution in a changing world[J].Nat Rev Microbiol, 2016, 14(7):421-33
|
| [8] |
KAPIKIAN A Z, WYATT R G, DOLIN R, et al.Visualization by Immune Electron-Microscopy of a 27-Nm Particle Associated with Acute Infectious Nonbacterial Gastroenteritis[J].Journal of Virology, 1972, 10(5):1075-81
|
| [9] |
KATAYAMA K, MURAKAMI K, SHARP T M, et al.Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA[J].Proc Natl Acad Sci U S A, 2014, 111(38):E4043-52
|
| [10] |
YI Z, PAN T, WU X, et al.Hepatitis C virus co-opts Ras-GTPase-activating protein-binding protein 1 for its genome replication[J].J Virol, 2011, 85(14):6996-7004
|
| [11] |
JONES M K, WATANABE M, ZHU S, et al.Enteric bacteria promote human and mouse norovirus infection of B cells[J].Science, 2014, 346(6210):755-9
|
| [12] |
ESTES M K, ETTAYEBI K, TENGE V R, et al.Human Norovirus Cultivation in Nontransformed Stem Cell-Derived Human Intestinal Enteroid Cultures: Success and Challenges[J].Viruses, 2019, 11(7):-
|
| [13] |
VAN DYCKE J, NY A, CONCEICAO-NETO N, et al.A robust human norovirus replication model in zebrafish larvae[J].PLoS Pathog, 2019, 15(9):e1008009-
|
|
|
|
