Rotavirus Infection and Cytopathogenesis in Human Biliary Organoids Potentially Recapitulate Biliary Atresia Development

Abstract

Biliary atresia (BA) is a neonatal liver disease characterized by progressive fibroinflammatory obliteration of both intrahepatic and extrahepatic bile ducts. The etiologies of BA remain largely unknown, but rotavirus infection has been implicated at least for a subset of patients, and this causal relation has been well demonstrated in mouse models. In this study, we aim to further consolidate this evidence in human biliary organoids. We obtained seven batches of human biliary organoids cultured from fetal liver, adult liver, and bile duct tissues. We found that these organoids are highly susceptible and support the full life cycle of rotavirus infection in three-dimensional culture. The robust infection triggers active virus-host interactions, including interferon-based host defense mechanisms and injury responses. We have observed direct cytopathogenesis in organoids upon rotavirus infection, which may partially recapitulate the development of BA. Importantly, we have demonstrated the efficacy of mycophenolic acid and interferon alpha but not ribavirin in inhibiting rotavirus in biliary organoids. Furthermore, neutralizing antibody targeting rotavirus VP7 protein effectively inhibits infection in organoids. Thus, we have substantiated the causal evidence of rotavirus inducing BA in humans and provided potential strategies to combat the disease.IMPORTANCE There is substantial evidence indicating the possible involvement of rotavirus in biliary atresia (BA) development, at least in a subset of patients, but concrete proof remains lacking. In a mouse model, it has been well demonstrated that rotavirus can infect the biliary epithelium to cause biliary inflammation and obstruction, representing the pathogenesis of BA in humans. By using recently developed organoids technology, we now have demonstrated that human biliary organoids are susceptible to rotavirus infection, and this provokes active virus-host interactions and causes severe cytopathogenesis. Thus, our model recapitulates some essential aspects of BA development. Furthermore, we have demonstrated that antiviral drugs and neutralizing antibodies are capable of counteracting the infection and BA-like morphological changes, suggesting their potential for mitigating BA in patients.

Keywords: biliary atresia; human organoids; rotavirus infection.

FIG 1

Characterizing rotavirus infection in human biliary organoids. (A) Dynamics of cellular viral RNA levels upon inoculation of SA11 rotavirus at different time points postinoculation. The level at 1 h postinoculation was set as 1. Three batches of human fetal liver organoids (FLOs), two adult liver organoids (LiOs), and two bile duct organoids (BDOs) were tested. Symbols and colors as for panel B. (B) Expression of rotavirus VP4 protein in the organoids determined by Western blotting. (C) Inoculation of human intestinal Caco2 cell line with supernatant from rotavirus-infected organoids for 48 h. Relative cellular viral RNA levels were quantified. (D) TCID₅₀ of five batches of rotavirus-infected organoids at 48 h postinoculation compared with the basal level at incubation. Organoids after inoculation were thoroughly washed to remove free viruses and subjected to repeated freezing and thawing to harvest the attached and entered rotaviruses. The total amounts of rotaviruses in organoids incubated for 48 h were harvested by repeated freezing and thawing of the entire well. (E) Volcano plots of differentially expressed genes in rotavirus-infected (for 48 h) compared to uninfected fetal liver organoids. (F) Gene ontology (GO) enrichment analysis of differentially expressed genes. All data are presented as means ± standard errors of the means (SEMs). For each organoid batch, experiments were repeated 3 to 6 times. *, P < 0.05; **, P < 0.01 by Mann-Whitney test.

FIG 2

Cytopathogenesis of rotavirus-infected human biliary organoids and efficacy of antiviral treatment/neutralizing antibody. (A) Organoids from 50 μm to 150 μm in diameter were selected to capture images. Optical microscopy images of infected and uninfected organoids (top). Fluorescence staining of dead cells (PI; red), live cells (calcein; green), and nuclei (Hoechst; blue) (middle). Confocal immunostaining of rotavirus structural protein VP6 (red), epithelial cell adhesion molecule (Epcam; green), and nuclei (blue) (bottom). These are representative images of one FLO batch from the tested seven biliary organoid batches. (B) Quantitative analysis of the percentage of deteriorated organoids with or without rotavirus infection at indicated time points and calculated based on LIVE/DEAD cell staining (A, middle). (C) The inhibitory activities of neutralizing monoclonal antibody HS-1 against rotavirus infection in three representative batches of biliary organoids. (D) The effects of the broad-spectrum antiviral drugs on rotavirus in biliary organoids. Rib, ribavirin; MPA, mycophenolic acid; IFN-α, interferon alpha. All data are presented as means ± SEMs. For each organoid batch, experiments were repeated 3 to 6 times. NS, not significant; **, P < 0.01 by Mann-Whitney test.