Elucidating the Pathogenesis of Chronic Norovirus in Inborn Errors of Immunity: Lessons from Single-Cell Transcriptomics of Biopsies and Patient-Derived Organoids

Chronic norovirus infection (CNI) causes significant morbidity and mortality in immunocompromised patients, including those with inborn errors of immunity (IEI). Gastrointestinal samples from patients with CNI demonstrate mild histopathologic changes. No animal model for human norovirus infection is routinely accessible, and norovirus cannot be passaged in traditional cell culture. In this study, we sought to identify the cell types permissive for human norovirus infection and to immunophenotype chronic norovirus-infected gastrointestinal tissue. Two patients with CNI and immunodeficiency underwent traditional clinical immunophenotyping and gastrointestinal biopsies. Immunodeficiency diagnoses were Good’s syndrome with severe T cell lymphopenia in patient 1 and NF-Kβ2 loss of function (LOF)/gain of function (GOF) disease in patient 2. Single-cell RNA-seq (scRNA-seq) was performed on patient duodenal biopsies as well as patient-derived enteroids. Patient datasets were compared with healthy individuals or norovirus-negative patients with environmental enteropathy from public databases. scRNA-seq of biopsy samples showed that late enterocytes contained the highest proportion of norovirus RNA; norovirus RNA was also present in M2 macrophages and enteroendocrine cells. Non-epithelial cells were present, including CD8, CD4, and γδ T cells, with a higher number of T cells bearing exhaustion markers, including CD160, TIGHT, and CXCL13, as compared to controls. Cells bearing B cell markers were absent in both patients. Furthermore, Rho GTPase, type I/II interferon, CXCL10, CXCL11, CXCL9, and PLA2G7 were all upregulated in patient samples. These tissue-based assays recapitulate findings from peripheral blood phenotyping, including absence of B cells and elevated systemic CXCL9 (range 5,088–6,987 pg/mL; upper limit of normal [ULN] 647 pg/mL). Additionally, CXCL9 staining was elevated in tissue via RNA-scope. scRNA-seq of patient-derived duodenal enteroids also showed upregulation of type I and type II interferon, as well as multiple cytokines, including CXCL10, CXCL11, and CX3CL1, especially within late enterocytes. Within the enteroids, late enterocyte cells supported norovirus replication. M2 macrophages differentiated from healthy donor peripheral blood mononuclear cells (PBMCs) incorporated viral particles, initiated viral replication and capsid protein synthesis, and released new viral RNAs/virions in vitro, suggesting an important role for tissue-based macrophages. Understanding the tissue-based immune dysregulation induced by chronic norovirus infection may help identify novel treatment strategies as well as tissue-based diagnostic targets and immunophenotyping.