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.

g Human norovirus is the leading cause of epidemic and sporadic acute gastroenteritis. Since no cell culture method for human norovirus exists, cultivable surrogate viruses (CSV), including feline calicivirus (FCV), murine norovirus (MNV), porcine enteric calicivirus (PEC), and Tulane virus (TuV), have been used to study responses to inactivation and disinfection methods. We compared the levels of reduction in infectivities of CSV and Aichi virus (AiV) after exposure to extreme pHs, 56°C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP), using the same matrix and identical test parameters for all viruses, as well as the reduction of human norovirus RNA levels under these conditions. At pH 2, FCV was inactivated by 6 log 10 units, whereas MNV, TuV, and AiV were resistant. All CSV were completely inactivated at 56°C within 20 min. MNV was inactivated 5 log 10 units by alcohols, in contrast to 2 and 3 log 10 units for FCV and PEC, respectively. TuV and AiV were relatively insensitive to alcohols. FCV was reduced 5 log 10 units by 1,000 ppm chlorine, in contrast to 1 log 10 unit for the other CSV. All CSV except FCV, when dried on stainless steel surfaces, were insensitive to 200 ppm chlorine. HHP completely inactivated FCV, MNV, and PEC at >300 MPa, and TuV at 600 MPa, while AiV was completely resistant to HHP up to 800 MPa. By reverse transcription-quantitative PCR (RT-qPCR), genogroup I (GI) noroviruses were more sensitive than GII noroviruses to alcohols, chlorine, and HHP. Although inactivation profiles were variable for each treatment, TuV and MNV were the most resistant CSV overall and therefore are the best candidates for studying the public health outcomes of norovirus infections.

Human noroviruses (HunoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HunoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HunoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-sydney HunoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HunoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. analysis of infection or attachment samples, including rna extraction and rt-qpcr, requires ~6 h.