New insights and enhanced human norovirus cultivation in human intestinal enteroids

Authors: ETTAYEBI, KHALIL - Baylor College Of Medicine; TENGE, VICTORIA - Baylor College Of Medicine; CORTES-PENFIELD, NICOLAS - Baylor College Of Medicine; CRAWFORD, SUE - Baylor College Of Medicine; NEILL, FREDRICK - Baylor College Of Medicine; ZENG, XI-LEI - Baylor College Of Medicine; YO, XIAOMIN - Baylor College Of Medicine; AYYAR, VIJAYALAKSMI - Baylor College Of Medicine; Burrin, Douglas - Doug; RAMANI, SASIREKHA - Baylor College Of Medicine; ATMAR, ROBERT - Baylor College Of Medicine; ESTES, MARY - Baylor College Of Medicine

Submitted to: American Society for Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2021
Publication Date: 1/27/2021
Citation: Ettayebi, K., Tenge, V., Cortes-Penfield, N., Crawford, S., Neill, F., Zeng, X., Yo, X., Ayyar, V., Burrin, D.G., Ramani, S., Atmar, R., Estes, M. 2021. New insights and enhanced human norovirus cultivation in human intestinal enteroids . American Society for Microbiology. 6(1):e01136-20. https://doi.org/10.1128/mSphere.01136-20.
DOI: https://doi.org/10.1128/mSphere.01136-20

Interpretive Summary: Human noroviruses are a leading viral cause of acute diarrhea world-wide. Noroviruses, also known as the cruise ship viruses. We recently developed a new experimental approach to examine how norovirus infect the intestine by using human intestinal biopsies to grow mini-guts, also called enteroids, in cell culture that replicate what happens in the human gut. In this report, we studied how different culture media and what region of the gut is used to make the mini-guts influences the infection rate of various norovirus strains. The results show that culturing mini-guts with media containing key factors from a novel cell line and media obtained commercially can promote robust growth of norovirus strains. The study also found the mini-guts made from patient biopsies taken from the upper gastro-intestinal (GI) tract allow norovirus growth, but those derived from the colon do not. These discoveries will give researchers important new information to improve the use of using mini-gut cultures to study norovirus infection in the human gut.

Technical Abstract: Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide. We previously demonstrated human intestinal stem cell-derived enteroids (HIEs) support cultivation of several HuNoV strains. However, HIEs did not support virus replication from every HuNoV-positive stool sample, which led us to test and optimize new medium conditions, identify characteristics of stool samples that allow replication, and evaluate consistency of replication over time. Optimization of our HIE-HuNoV culture system has shown the following: (i) a new HIE culture medium made with conditioned medium from a single cell line and commercial media promotes robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture medium made with conditioned media from 3 separate cell lines; (ii) GI.1, 11 GII genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.12, GII.13, GII.14, and GII.17), and six GII.4 variants can be cultivated in HIEs; (iii) successful replication is more likely with virus in stools with higher virus titers; (iv) GII.4_Sydney_2012 virus replication was reproducible over 3 years; and (v) HuNoV infection is restricted to the small intestine, based on replication of two viral strains in duodenal and ileal HIEs, but not colonoids, from two susceptible donors. These results improve the HIE culture system for HuNoV replication. Use of HIEs by several laboratories worldwide to study the molecular mechanisms that regulate HuNoV replication confirms the usefulness of this culture system, and our optimized methods for virus replication will advance the development of effective therapies and methods for virus control. Human noroviruses (HuNoVs) are highly contagious and cause acute and sporadic diarrheal illness in all age groups. In addition, chronic infections occur in immunocompromised cancer and transplant patients. These viruses are antigenically and genetically diverse, and there are strain-specific differences in binding to cellular attachment factors. In addition, new discoveries are being made on strain-specific differences in virus entry and replication and the epithelial cell response to infection in human intestinal enteroids. Human intestinal enteroids are a biologically relevant model to study HuNoVs; however, not all strains can be cultivated at this time. A complete understanding of HuNoV biology thus requires cultivation conditions that will allow the replication of multiple strains. We report optimization of HuNoV cultivation in human intestinal enteroid cultures to increase the numbers of cultivatable strains and the magnitude of replication, which is critical for testing antivirals, neutralizing antibodies, and methods of virus inactivation.