Advances in understanding of the innate immune response to human norovirus infection using organoid models

Abstract

Norovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and the most frequent cause of foodborne illness in the United States. There is no specific treatment for norovirus infections and therapeutic interventions are based on alleviating symptoms and limiting viral transmission. The immune response to norovirus is not completely understood and mechanistic studies have been hindered by lack of a robust cell culture system. In recent years, the human intestinal enteroid/human intestinal organoid system (HIE/HIO) has enabled successful human norovirus replication. Cells derived from HIE have also successfully been subjected to genetic manipulation using viral vectors as well as CRISPR/Cas9 technology, thereby allowing studies to identify antiviral signaling pathways important in controlling norovirus infection. RNA sequencing using HIE cells has been used to investigate the transcriptional landscape during norovirus infection and to identify antiviral genes important in infection. Other cell culture platforms such as the microfluidics-based gut-on-chip technology in combination with the HIE/HIO system also have the potential to address fundamental questions on innate immunity to human norovirus. In this review, we highlight the recent advances in understanding the innate immune response to human norovirus infections in the HIE system, including the application of advanced molecular technologies that have become available in recent years such as the CRISPR/Cas9 and RNA sequencing, as well as the potential application of single cell transcriptomics, viral proteomics, and gut-on-a-chip technology to further elucidate innate immunity to norovirus.

Keywords: enteroids; innate immunity; norovirus.

Fig. 1.

The antiviral response in norovirus-infected cells. Norovirus infection induces an antiviral response that restricts virus replication. a. Human norovirus attachment is facilitated by human blood group antigens (HBGA) and a receptor that is yet to be identified. In mice the receptor is CD300lf [66]. b. Viral entry results in sensing of the virus and viral components by molecular sensors. The molecular sensors that are triggered during human norovirus infection have not been identified, however human norovirus virus-like particles (VLPs) have been shown to trigger TLR2 and TLR5 (70). MDA5 and RIG-I are also involved in the response against human norovirus [102] and TLR7 has been shown to trigger an antiviral response that protects against murine norovirus (MNV) [103]. c. Virus sensing results in activation of transcription factors including NFkB [103] and interferon regulatory factors IRF1(102), IRF3 and IRF7(3) which facilitate transcription of genes encoding type I, type II and type III interferon. d. Interferon is secreted and engages corresponding receptors on the cell surface, resulting in phosphorylation and activation of STAT1/2 through the JAK/STAT pathway [55]. e. STAT1/2 translocate to the nucleus and facilitate upregulation of hundreds of antiviral interferon stimulated genes (ISGs). f. The proteins encoded by antiviral ISGs restrict virus replication. This figure summarizes the immune components involved in the antiviral response to norovirus infection and includes findings from human norovirus infection as well as its surrogates: ^¥human norovirus infection in human intestinal enteroids, ^*murine norovirus, ^# human norovirus virus like particles (VLPs), ^§human norovirus replicon. Created with BioRender.com (accessed on 12 October 2021).

Fig. 2.

Application of human intestinal enteroids/organoids (HIE/HIO) in understanding the immune response to human norovirus infection. HIE/HIO can be applied in multiple ways to investigate the immune response to norovirus infection and determine host antiviral factors that restrict norovirus replication. HIE and HIO are derived either from adult intestinal biopsies, or adult induced pluripotent cells (iPSCs). Once HIE/HIO are propagated and differentiated, they can be infected with human norovirus. The cells can also be genetically manipulated using CRISPR/Cas9 or viral vectors to create mutant cell lines that can also be used in norovirus studies. Cells and RNA derived from infected HIE/HIO can be analysed for transcriptional changes using RNA sequencing. Similarly, the cells can also be subjected to proteomics-based analyses to investigate effects of norovirus infection on expression. Another potential application of HIE/HIO is measuring metabolites during norovirus infection using gut-on a chip technology. Created with BioRender.com (accessed on 12 October 2021).