Investigate virus-host interaction of enterovirus and coronavirus in human intestinal organoids

Abstract

Enteric infections of enterovirus and coronavirus represent a serious and constant threat for humans. The immortalized cell lines conventionally used to study the virus-host interactions are unable to model the cellular diversity and complex functionality of human intestinal epithelium. Human intestinal organoids can morphylogically and functionally simulate the native human intestinal epithelium, and have become a physiological-active and robust ex vivo model to study virus-host interaction in human intestinal cells. Enteroviruses, such as EV-A71 and CVA16, mainly infect the human gastrointestinal tract. Human coronaviruses, including SARS-CoV and SARS-CoV-2, have been variably associated with gastrointestinal symptoms. We aimed to optimize human intestinal organoids and hypothesized that these optimized intestinal organoids could recapitulate enteric infections of enterovirus and coronavirus. We demonstrated that the optimized human intestinal organoids enable better simulation of the native human intestinal epithelium, and they are significantly more susceptible to EV-A71 than CVA16. Higher replication of EV-A71 than CVA16 in intestinal organoids triggers a more vigorous cellular response. In contrast, more robust propagation of SARS-CoV triggers a minimal cellular response, whereas SARS-CoV-2 exhibits lower replication capacity but elicits a moderate cellular response. The disparate profile of virus-host interaction of enteroviruses and coronaviruses in human intestinal organoids may unravel the cellular basis of distinct pathogenicity of these viral pathogens. EV-A71 is the major causative agent of hand-foot-and-mouth diseases. To identify potential host kinases involved in the EV-A71 replication, we performed a kinase library screening in RD cells and human intestinal organoids simultaneously. Firstly, we identified several kinase inhibitors that substantially abolished viral growth. Among the top hits, we found that the Rock inhibitor efficiently suppressed the replication of EV-A71 in both RD cells and human intestinal organoids in a dose-dependent manner. Furthermore, we demonstrated that genetic depletion of Rock1, but not Rock2, significantly restricted the viral replication, indicating that Rock1 is required for EV-A71 replication. Thus, our findings suggest that Rock1 is a novel host factor for EV-A71 replication. Moreover, Rock inhibitors could be developed as antivirals against EV-A71. Plenty of studies in MERS patients suggested that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of MERS-CoV. We sought to identify pattern recognition receptor (PRR) signaling pathway(s) that mediates the exuberant inflammation in MERS-CoV-infected human macrophage. We found that disrupting RLR or CLR signaling, especially at the adaptor level, largely dampened the induction of proinflammatory cytokines and chemokines by MERS-CoV. Since the role of RLR in immune activation in coronavirus infections has been studied extensively, we charactized the interplay of CLR signaling and MERS-CoV. MERS-CoV replication significantly upregulated CLR receptor Mincle. The essential role of Mincle for MERS-CoV-triggered proinflammatory response was demonstrated based on the results of antibody blockage, siRNA depletion of Mincle and its adaptor Syk, and Syk pharmacological inhibition. Overall, the knowledge obtained from this study will facilitate the understanding of virus-host interplay and provide a scientific basis for the implementation of effective therapeutic strategies to combat human infections of enterovirus and novel coronavirus.