Single-cell transcriptomic analysis of duodenal biopsies and enteroids from immunocompromised patients with chronic norovirus infection

Yuki Matsushima, Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health

Ryoka Ishiyama1, Natthawan Chaimongkol1, Jessica Durkee-Shock2, Karenna Barton2, Jeffrey I Cohen2, Michael D Keller3, 4, Alison Han5, Bianca M Nagata6, Derron A Alves6, Eunice Chan1, Sadhana S. Sagar1, Stanislav V. Sosnovtsev1, and Kim Y. Green1. 1 Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, USA; 2 Medical Virology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, USA; 3 Center for Cancer and Immunology Research, Children's National Hospital, Washington, District of Columbia, USA; 4 Division of Allergy and Immunology, Children's National Hospital, Washington, District of Columbia, USA; 5 Laboratory of Infectious Diseases Clinical Studies Unit, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, USA; 6 Infectious Disease Pathogenesis Section, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Rockville, Maryland, USA.

Entry and Replication

To identify and characterize cell types permissive to human norovirus infection, we conducted single-cell RNA sequencing (scRNAseq) of duodenal biopsies and enteroids derived from two patients with T and B cell immunodeficiencies and chronic norovirus infection. The scRNAseq dataset was compared with that available in public databases derived from healthy individuals or norovirus negative patients with environmental enteropathy as controls. Unsupervised clustering analysis showed that the epithelial cells from duodenal biopsies in our two patients included enterocytes at distinct stages of differentiation, such as early, intermediate and late. Specialized epithelial cell types, including goblet, enteroendocrine, tuft, paneth and BEST4 cells were also present in the biopsies. Late enterocytes presented the highest proportion of epithelial cells containing norovirus RNA. The non-epithelial cells included CD8, CD4 and gamma-delta T cells, with a higher number of T cells bearing exhaustion markers as compared to controls. Cells bearing B cell markers were absent in both patients, consistent with their diagnosed immunodeficiencies. We identified multiple cell types with specific marker genes in the myeloid immune cell cluster, with activated macrophage presenting the highest level of norovirus RNA in the non-epithelial cells. A comparative analysis between duodenal biopsies and enteroids revealed clustering differences among stem cells and enterocytes. Understanding the cell types that support human norovirus replication may lead to improvement of in vitro cell culture systems for the virus and provide insight into mechanisms of viral replication and persistence.