Human Norovirus Replication in Human Intestinal Enteroids as Model to Evaluate Virus Inactivation

Abstract

Human noroviruses are a leading cause of epidemic and endemic acute gastroenteritis worldwide and a leading cause of foodborne illness in the United States. Recently, human intestinal enteroids (HIEs) derived from human small intestinal tissue have been shown to support human norovirus replication. We implemented the HIE system in our laboratory and tested the effect of chlorine and alcohols on human norovirus infectivity. Successful replication was observed for 6 norovirus GII genotypes and was dependent on viral load and genotype of the inoculum. GII.4 viruses had higher replication levels than other genotypes. Regardless of concentration or exposure time, alcohols slightly reduced, but did not completely inactivate, human norovirus. In contrast, complete inactivation of the 3 GII.4 viruses occurred at concentrations as low as 50 ppm of chlorine. Taken together, our data confirm the successful replication of human noroviruses in HIEs and their utility as tools to study norovirus inactivation strategies.

Use of Human Intestinal Enteroids to Detect Human Norovirus Infectivity

“…We hypothesize that a substantial proportion of asymptomatic patients are shedding infectious norovirus and that their role in spreading the virus merits our attention. We have shown that high levels of norovirus replication can be achieved from fecal samples of adults, not just young children, in HIE cultures ( 11 ). …”

“…Second, virus culture systems in cell lines generally lack sensitivity ( 12 ), and that of HIE remains unknown. However, it is probably not optimal because input genome equivalents for norovirus to achieve replication are not extremely low (50% infectious dose 4.4 × 10 2 to 2.1 × 10 3 copies/well) ( 3 , 11 ). It is possible that samples with C t >30 might still contain infectious virus and that low amounts of replicating norovirus would only be detected with further serial propagation of the virus.…”

“…It is possible that samples with C t >30 might still contain infectious virus and that low amounts of replicating norovirus would only be detected with further serial propagation of the virus. Third, norovirus replication varies between samples and virus genotypes in HIE culture ( 11 ). …”

Human Norovirus Neutralized by a Monoclonal Antibody Targeting the HBGA Pocket

“…Recently, a system utilizing human intestinal enteroids (HIE) was reported to support replication of human norovirus (10). Moderate replication levels could be enhanced with addition of bile acids, however the replication success was still strongly dependent on the particular stool sample (28). This cell culture system highlighted the requirement of HBGAs on the surface of permissive cells and showed that HBGA blocking sera can effectively neutralize norovirus.…”

“…This cell culture system highlighted the requirement of HBGAs on the surface of permissive cells and showed that HBGA blocking sera can effectively neutralize norovirus. Importantly, the HIE system was used to evaluate the neutralizing properties of 25 different GII.4 specific IgG and IgA isolated from a human donor (28). Several antibodies were able to neutralize human norovirus and were predicted to bind to the P domain, however the precise binding sites were not yet determined.…”