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bioRxiv · 10.1101/2020.11.12.380022

New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids

Ettayebi, K.·Tenge, V. R.·Cortes-Penfield, N. W.·Crawford, S. E.·Neill, F. H.·Zeng, X.-L.·Yu, X.·Ayyar, V. B.·Burrin, D.·Ramani, S.·Atmar, R. L.·Estes, M. K.
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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 media 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 that: 1) A new HIE culture media made with conditioned medium from a single cell line and commercial media promote robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture media made with conditioned media from 3 separate cell lines; 2) GI.1, eleven 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; 3) successful replication is more likely with virus in stools with higher virus titers; 4) GII.4_Sydney_2012 virus replication was reproducible over three years; and 5) HuNoV infection is restricted to the small intestine, based on replication in duodenal and ileal HIEs but not colonoids from the same 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. ImportanceHuman 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.

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Ettayebi, K., Tenge, V. R., Cortes-Penfield, N. W., Crawford, S. E., Neill, F. H., Zeng, X.-L., Yu, X., Ayyar, V. B., Burrin, D., Ramani, S., Atmar, R. L., Estes, M. K.. 2020-11-12. New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids. https://doi.org/10.1101/2020.11.12.380022

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