Targeted Profiling of Immunological Genes during Norovirus Replication in Human Intestinal Enteroids

Abstract

Norovirus is the leading cause of acute gastroenteritis worldwide. The pathogenesis of norovirus and the induced immune response remain poorly understood due to the lack of a robust virus culture system. The monolayers of two secretor-positive Chinese human intestinal enteroid (HIE) lines were challenged with two norovirus pandemic GII.4 Sydney strains. Norovirus RNA replication in supernatants and cell lysates were quantified by RT-qPCR. RNA expression levels of immune-related genes were profiled using PCR arrays. The secreted protein levels of shortlisted upregulated genes were measured in supernatants using analyte-specific enzyme-linked immunosorbent assay (ELISA). Productive norovirus replications were achieved in three (75%) out of four inoculations. The two most upregulated immune-related genes were CXCL10 (93-folds) and IFI44L (580-folds). Gene expressions of CXCL10 and IFI44L were positively correlated with the level of norovirus RNA replication (CXCL10: Spearman’s r = 0.779, p < 0.05; IFI44L: r = 0.881, p < 0.01). The higher level of secreted CXCL10 and IFI44L proteins confirmed their elevated gene expression. The two genes have been reported to be upregulated in norovirus volunteer challenges and natural human infections by other viruses. Our data suggested that HIE could mimic the innate immune response elicited in natural norovirus infection and, therefore, could serve as an experimental model for future virus-host interaction and antiviral studies.

Norovirus: Facts and Reflections from Past, Present, and Future

“…The detection of viral non-structural proteins in enterocytes of human adults' biopsies [97], and the presence of viral non-sense RNA in intestinal enteroendocrine cells from pediatric intestinal biopsies [98], suggest that HuNoV replicates in intestinal cells during infection, which is supported by its ability to replicate in human intestinal enteroids (HIE) [57,99]. While replication of HNoV in B cell lines has been shown [100,101], there is lack of evidence of in vivo replication in immune cells [100].…”

“…In humans, both a proinflammatory and a regulatory cytokine response is seen during the acute phase [106], which may be related to symptomatology [107]. HNoV replication in HIEs systems have provided evidence of the importance of innate immunity in its pathogenesis; while HNoV triggers a chemokine (CXCL10) [99] and IFN-stimulated genes with a predominantly type III IFN response; however, there is a strain-dependent sensibility to endogenous IFN which can explain the presence of globally expanded strains more resistant to innate-immunity signaling (such as GII.4) [108]. Adaptive immune response to MNoV requires both humoral and cellular components to be effective [109].…”

Dasabuvir inhibits human norovirus infection in human intestinal enteroids

“…Recently, several HuNoV successive cultivation models employing a human B cell line (Jones et al, 2014), tissue stem cell-derived human intestinal enteroids (HIEs) (Ettayebi et al, 2016), human induced pluripotent stem cell-derived intestinal organoids (Sato et al, 2019), and zebrafish larvae (Van Dycke et al, 2019) have been developed. The stem cell-derived HIE system is currently used by researchers worldwide to study HuNoV biology and inactivation strategies (Alvarado et al, 2018; Chan et al, 2021; Costantini et al, 2018; Ettayebi et al, 2021; Hosmillo et al, 2020; Lin et al, 2020; Murakami et al, 2020), although, to our knowledge, compound screens for identifying HuNoV antiviral agents have not been reported.…”

Advancements in Human Norovirus Cultivation in Human Intestinal Enteroids

“…This development overcame a five-decade barrier in laboratory models for HuNoVs, opening new avenues for investigating HuNoV biology. Since the establishment of the HIE system, significant advancements have been made in unraveling various aspects of HuNoV biology, including strain-specific differences in replication, methods of virus inactivation, innate immune responses, antiviral susceptibility, and characterization of neutralizing antibodies (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Here, we report further advancements in the HIE culture system by testing HuNoV infection in multiple unique HIE lines and expanding the profile of cultivatable HuNoV strains.…”