Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids

Abstract

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2. An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.

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

“…Therefore, both innate immunity and IFNs were the most well-studied responses based on animal models, which helped in selecting appropriate PCR array pathways. Two recent studies using RNA-seq on HIE have shown that interferon responses were mounted upon human GII.4 norovirus infection [ 37 , 38 ], which has strengthened our choice of PCR array pathways.…”

“…In this UK study, the authors tested HuNoV in duodenal and terminal ileal organoids derived from ethnic Caucasians and performed RNA-seq in terminal ileal organoids only. Another study that performed transcriptomic analyses also reported a statistically significant increase in the expression of IFI44L at 24 hpi in Caucasians jejunal enteroids inoculated with GII.4 norovirus [ 37 ]. By comparing our data with the two studies, it likely suggested that HuNoV may induce a general immune response in HIEs irrespective of its position within the gastrointestinal tract and host ethnicity.…”

“…In a study that performed staining on duodenal, jejunal and ileal biopsies obtained from HuNoV-positive patients, it was shown that viral protein 1 was detected in different parts of the small intestine, indicating that all segments of the small intestine support HuNoV replication [ 35 ]. Since a majority of the studies uses jejunal- and ileal-derived HIE [ 34 , 36 , 37 , 38 ], we therefore derived duodenal HIE to investigate the difference between infection sites. Using immune response as a parameter, we further investigated the correlation between immune response and HuNoV replication in HIE and evaluated the robustness of the HIE model by comparing our observations with HuNoV naturally infected cases in the literature and other studies.…”

New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids

“…The lack of a reproducible culture system for HuNoVs was a major barrier to understanding virus biology including mechanisms of replication, inactivation, neutralization, and vaccine development for approximately five decades (11,12). This problem was overcome with the successful cultivation of multiple HuNoV strains in enterocytes in human intestinal stem cell-derived, nontransformed enteroid (HIE) monolayer cultures (12)(13)(14)(15)(16)(17)(18)(19)(20). Previous studies showed replication of GI.1 and six GII genotypes, including four GII.4 variants, in this ex vivo system, and virus replication in HIEs mimics epidemiological differences in host susceptibility based on genetic differences in expression of histo-blood group antigens (HBGAs) as defined by a person's secretor status (11-13, 21, 22).…”

“…7). (12,18,21,24). We previously defined the BA-mediated mechanism for GII.3 replication involves virus uptake mediated by dynamic and rapid BAmediated cellular endolysosomal dynamic changes and cellular ceramide (24).…”