Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape

Abstract

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLBtype and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT PLOS Pathogens | https://doi.and ALSAC to SSC.; NIH R21 NS101371 to DW; Wellcome Trust: Ref 207498/Z/ signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions. Author summaryHuman astroviruses (HAstV) are understudied positive-strand RNA viruses that typically cause gastroenteritis mostly in children and the elderly, but more recent studies also implicate them in neurological disease in immunocompromised patients. To better understand these viruses, a physiologically relevant cell culture model that supports growth of all clades of HAstV would be highly beneficial. Herein, we demonstrated robust infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts from different patients and intestinal regions, making HIE a valuable model to study HAstV biology. Using this system, we identify for the first time that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes. Analysis of the antiviral host response to infection demonstrated that HIE respond to infection with a type I and III interferon response. This response reduced HAstV replication and when blocked resulted in increased infection. Establis...

Infectious Norovirus Is Chronically Shed by Immunocompromised Pediatric Hosts

“…Human intestinal enteroids derived from fetal ileum were obtained from the University of Michigan Medical School Translational Tissue Modeling Laboratory. Briefly, the 3-dimensional (3D) organoids were suspended in Corning Matrigel (Tewksbury, MA, USA) and maintained in complete L-WRN medium as described [ 33 ]. Prior to infection, 3D enteroids were dissociated into single cells and seeded as 2D monolayers in 48-well plates that were coated with human collagen type IV at a density of 1 × 10 5 cells per well.…”

Enteric Viral Co-Infections: Pathogenesis and Perspective

“…HIOs support the replication of clinical isolates of RV, and HIEs have been used to study host restriction and innate antiviral responses to RV [206,207]. Multiple human NoV strains have been successfully cultivated in HIEs derived from human intestinal biopsies [208], as have human AstV [53]. HIE models have also been pivotal in investigating antiviral signaling responses to enteroviruses and coxsackieviruses [209,210].…”

Somatic cell-derived organoids as prototypes of human epithelial tissues and diseases

“…Organoids have further contributed host cells for a range of viruses, such as influenza virus [122][123][124] , respiratory syncytial virus 16 , human norovirus 125 , human rotavirus [126][127][128] , enteroviruses 129 , human adenovirus 130 , BK virus 31 , human papillomavirus 15 , herpes simplex virus 15 , human astrovirus 131 and Middle East respiratory syndrome coronavirus (MERS-CoV) 132 . Of note, some human norovirus strains required bile supplementation for their efficient replication in human small intestinal organoids 125 .…”