Host and environmental modulators of norovirus infection

Abstract

Human noroviruses (HNoV) are the leading cause of non-bacterial gastroenteritis worldwide. Despite the significant disease and economic burden, currently no licensed vaccines or antivirals are available against norovirus infection. Progress in understanding norovirus biology has long been hindered by the inability to efficiently cultivate HNoV. This limitation was overcome with the development of the human intestinal enteroid (HIE) platform, which enables infection with clinical HNoV isolates in a physiologically relevant system that recapitulates the cellular diversity and the architecture of the human gut. This dissertation investigates how host and environmental factors influence HNoV infection in the 3DHIE. A robust and reproducible HNoV infection model in 3D-HIEs enabled detailed investigation of viral entry, replication, passaging, and virus-host interactions. From a laboratory owned biobank of diagnostic stool samples, four infectious HNoV isolates were identified, sequenced and genotyped. Through virological and biochemical approaches, glycans, divalent cations, bile acids, and polyamines were characterized as key host determinants that modulate HNoV infectivity, replication, and escape from antibody neutralization. The 3D-HIE platform was extended to also study infection of a clinical human rotavirus (HRoV) isolate and an established human influenza A virus (IAV) laboratory strain. Active fucosylation in 3D-HIEs was identified as a critical determinant for infection by both HNoV GII.4 and HRoV G3.P[8]eq.-like strain but not IAV. However, use of fucosylated surface glycans differed between HNoV and HRoV as identified by lectin competition. Exogenous fucose supplementation enhanced HNoV replication rather than competing with infection. Environmental ligands, specifically divalent cations and bile acids, were shown to shape norovirus infectivity and immune recognition. In both murine and human norovirus systems, magnesium ions and the bile glycochenodeoxycholic acid (GCDCA) were enhancing MNV and HNoV infections. Magnesium ions facilitated escape from neutralizing antibodies for two MNV strains and one GII.4 HNoV stool-isolate. For HNoV, magnesium was able to revert a loss in viral titers observed after storage of diluted stool isolates. Host-derived polyamines were identified as broad host dependency factors for norovirus infection. Depletion of polyamines strongly suppressed MNV replication, inhibited capsid protein synthesis, and prevented virus-induced cytopathic effect (CPE), in a reversible manner. Mechanistic studies linked polyamine metabolism, norovirus replication, and virus-induced CPE with PI3K/Akt signaling and cholesterol metabolism as downstream targets. The dependency on polyamines for both MNV and HNoV infections suggests a broad relevance and a novel target for antiviral therapy suited to pharmacologically target chronic norovirus disease. Collectively, this work advances understanding of the host and environmental determinants that promote norovirus infection by shaping viral entry, replication, infectivity, and neutralization escape. The identified host dependency factors, including fucosylated glycans and polyamines, emerge as critical regulators of infection and promising therapeutic targets, while the influence of environmental modulators on HNoV GII.4 infection and antibody escape provides important insights into norovirus biology and caveats for rational vaccine development.