Establishment of three human intestinal enteroid lines to study human norovirus infection
Refereed conference paper presented and published in conference proceedings


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AbstractBackground and Aim
Norovirus is the leading cause of acute gastroenteritis worldwide and is accounted for nearly one-fifth of all-cause cases annually. The pathogenesis of norovirus remains poorly understood due to the lack of a robust tissue culture system. Not until 2016, norovirus has finally been cultivated in an adult stem-cell derived three-dimensional human intestinal enteroid (HIE) system. Here, we aim to establish HIE lines to study human norovirus infection.

Method
HIEs were derived from duodenal biopsies obtained from consented donors via oesophago-gastro-duodenoscopy, followed by the isolation of Lgr5 intestinal stem cells at the crypts. The cells obtained were cultured in Matrigel. Complete medium with growth factors composed of appropriate proportion of Wnt3a, Noggin and R-spondin conditioned medium along with Y-27632 were used to maintain HIE. Saliva samples were collected for FUT2 A385T genotyping by a TaqMan-based allelic discrimination assay and for histo-blood group antigen phenotyping through an enzyme-linked immunosorbent assay to determine host secretor status.

Results
A total of three patient-derived HIE lines were established from duodenal biopsies. Two of them were typed as secretor, and one was typed as weak secretor, of which the two secretor lines were belonged to blood group A and AB respectively. The enteroid lines were successfully passaged for at least 15 times with approximately 50-100 enteroids per well and could grow up to 300 µm in size robustly. Cryopreserved stocks of HIE were made available before P6 for all lines and their viability remained high after thawing.

Conclusion
We have successfully established three HIE lines of different histo-blood groups. The susceptibility of the two secretor enteroid lines to infection by norovirus GII.4 Sydney strains will be evaluated in the future.

Acknowledgements
This work was supported in part by the Hong Kong Research Grants Council (to M.C.W.C.; reference number 14162217).
All Author(s) ListJenny C.M. Chan, Kirran N. Mohammad, Sunny Hei Wong, Martin Chi-Wai Chan
Name of ConferenceINFECTION 2019
Start Date of Conference10/06/2019
End Date of Conference11/06/2019
Place of ConferenceHong Kong
Country/Region of ConferenceHong Kong
Year2019
Month6
LanguagesEnglish-United Kingdom
Keywordsnorovirus, enteroids

Last updated on 2019-17-06 at 12:51