Based on current work, in vitro studies demonstrate that TV is an overall more conservative and suitable surrogate for the study of HuNoV physicochemical properties.

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Human norovirus (HuNoV) is the main cause of gastroenteritis worldwide, yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an interferon (IFN)-cured population of replicon-harboring HGT cells (HGT-Cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-Cured cells created an environment favoring norovirus replication. Microarrays were used to identify genes differentially regulated in HGT-NV and HGT-Cured compared to parental cells. We found that IFN lambda receptor (IFNLR1) expression was highly reduced in HGT-NV and HGT-Cured cells. While all three cell lines responded to exogenous IFN-β by inducing interferon-stimulated genes, HGT-NV and HGT-Cured cells failed to respond to exogenous IFN-λ. Methylation-sensitive PCR showed that an increased methylation of the IFNLR1 promoter and inhibition of DNA methyltransferase activity partially reactivated IFNLR1 expression in HGT-NV and HGT-Cured cells, indicating that host adaptation occurred via epigenetic reprogramming. Moreover, IFNLR1 ectopic expression rescued response to IFN-λ and restricted HuNoV replication in HGT-NV cells. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. This study unravels for the first time epigenetic reprogramming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signaling is able to control human norovirus replication. IMPORTANCE Noroviruses are one of the most widespread causes of gastroenteritis, yet no suitable therapeutics are available for their control. Moreover, to date, knowledge of the precise cellular processes that control the replication of the human norovirus remains ill defined. Recent work has highlighted the importance of type III interferon (IFN) responses in the restriction of viruses that infect the intestine. Here, we analyzed the adaptive changes required to support long-term replication of noroviruses in cell culture and found that the receptor for type III IFN is decreased in its expression. We confirmed that this decreased expression was driven by epigenetic modifications and that cells lacking the type III IFN receptor are more permissive for norovirus replication. This work provides new insights into key host-virus interactions required for the control of noroviruses and opens potential novel avenues for their therapeutic control.

Human enteric viruses have been identified as one of the predominant causative agents of food-borne illnesses in developed countries, and it is estimated that human norovirus accounts for a majority of these illnesses each year. Not all of these viruses can be cultured and hence relatively little is known about their pathogenesis and physicochemical properties. To overcome this, researchers have utilized different virus surrogates for the study of non-cultivable human enteric viruses. In this review, we discuss various methods utilized for the evaluation of the thermal stability of human enteric viruses, compare the results of these methods, and examine how researchers may move toward a single standard approach (i.e., temperatures, virus concentrations, volume/weight of matrices, etc.) for determining thermal inactivation profiles of human enteric viruses and their surrogates. Based on our review, we found that temperature, time of exposure, type of matrix, analysis type, type of heat application, and the concentration and volume of virus used in the experiments were highly variable across virus surrogates even for the same surrogates. Because of these differences-along with the inherent limitations of using surrogate viruses-comparison of these methods and how the results may be extrapolated to human enteric viruses is quite challenging. As a result, we discuss how researchers may move toward a single standard approach for determining thermal inactivation profiles of human enteric viruses and their surrogates.

The cGAS-STING pathway is central to the IFN response against DNA viruses. However, recent studies are increasingly demonstrating its role in the restriction of some RNA viruses. Here we show that the cGAS-STING pathway also contributes to the IFN response against noroviruses, positive-sense single-stranded RNA viruses that are now one of the most common causes of infectious gastroenteritis world-wide. We show a significant reduction in IFN-β induction and a corresponding increase in viral replication in norovirus-infected cells following STING inhibition, knockdown or deletion. Upstream of STING, we show that cells lacking either cGAS or IFI16 also have severely impaired IFN responses. Further, we demonstrate that immunostimulatory host genome-derived DNA, and to a lesser extent mitochondrial DNA, accumulate in the cytosol of norovirus-infected cells. And lastly, overexpression of the viral NS4 protein was sufficient to drive the accumulation of cytosolic DNA. Together, our data elucidate a role for cGAS, IFI16 and STING in the restriction of noroviruses, and demonstrate for the first time the utility of host genomic DNA as a damage-associated molecular pattern in cells infected with an RNA virus.

Human noroviruses (HuNoVs) are the leading cause of acute viral gastroenteritis worldwide. The persistence of HuNoV in the environment contributes significantly to its transmission to humans. Surrogate viruses are used to study HuNoV owing to the lack of a cell culture system for this virus. Here, the persistence of Tulane virus (TV) - a novel HuNoV surrogate - in surface water (SW) and groundwater (GW) as well as on acrylic-based solid (ABS) and stainless steel (SS) surfaces was investigated. After 28 days, TV remained stable in SW (<1 log10 reduction) but was reduced by ≥3.5 to 4 log10 in GW by day 21. TV had a higher rate of reduction on SS compared with ABS, with corresponding D values of 18.5 ± 0.34 and 13.1 ± 0.36 days, respectively. This is the first study to demonstrate the persistence of TV in environmental waters and on fomite surfaces.

Human norovirus (HuNoV) is the main cause of gastroenteritis worldwide yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an IFN-cured population of replicon-harboring HGT cells (HGT-cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-cured cells created an environment favouring norovirus replication. Microarray analysis was used to identify genes differentially regulated in HGT-NV and HGT-cured compared to parental HGT cells. We found that the IFN lambda receptor alpha (IFNLR1) expression was highly reduced in HGT-NV and HGT-cured cells. All three cell lines responded to exogenous IFN-β by inducing interferon stimulated genes (ISGs), however, HGT-NV and HGT-cured failed to respond to exogenous IFN-λ. Inhibition of DNA methyltransferase activity with 5-aza-2'-deoxycytidine partially reactivated IFNLR1 expression in HGT-NV and IFN-cured cells suggesting that host adaptation occurred via epigenetic reprogramming. In line with this, ectopic expression of the IFN-λ receptor alpha rescued HGT-NV and HGT-cured cells response to IFN-λ. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. This study unravels for the first time epigenetic reprogramming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signalling is able to control human norovirus replication.

No abstract