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

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.

Page 2 of 8 kindly provided by Dr. Calvin Kuo, Palo Alto, CA. Noggin-producing cells were kindly provided by Dr. Gijs van den Brink, University of Amsterdam, Netherlands. Complete media with and without growth factors (CMGF + and CMGF -, respectively), differentiation media, and Wnt3a-R-spondin-and Noggin -conditioned media were prepared as reported previously (1,2).
Jejunal HIE cultures (J2 or J3 lines) were grown as undifferentiated 3-dimensional (3D) cultures, as described previously (2) with minor modifications. Briefly, HIEs were recovered from liquid nitrogen (LN2), suspended in 20 L of Matrigel ® (40 crypts), plated in a single well of a 24-well plate, and grown as 3D cultures in CMGF + medium supplemented with 10 M Y-27632 (Sigma). We replaced the medium every 48 hours. After 7 days, highly dense 3D cultures were either split 1:2 and embedded in Matrigel, frozen in liquid nitrogen for further use, or dissociated into single cell suspension and plated as undifferentiated monolayers, as described previously (2) (Technical Appendix Figure 1).
For all infections, we washed undifferentiated HIEs with 0.5 mM EDTA in ice-cold PBS (without calcium chloride-magnesium chloride) and dissociated into single cell suspension with 0.05% trypsin/0.5 mM EDTA. We seeded 96 well plates with 1-2  10 5 cells/well to form monolayers, as described previously (2). After 24 hours, CMGF + supplemented with 10 M Y-27632 was replaced with differentiation medium, which was refreshed every 48 hours during 4 days.

Total RNA Isolation and Gene Expression Analysis
We isolated total RNA from differentiated and undifferentiated HIE monolayers using Applied Biosystems 7500 platform. Expression levels were normalized to GAPDH and foldchange of expression level was calculated using the comparative Ct method (2 -Ct ), as described previously (3). Heat maps were created using GraphPad Prism7.0 (GraphPad Software, La Jolla, CA, USA).

Infection Experiments and Viral Replication
We performed all infections in triplicate on 100% confluent 4-day-old differentiated HIE (J3 line) monolayers in 96-well plates, except when specified that J2 line was used. In some experiments, monolayers were pretreated with 1% sow bile included in the differentiation medium 48 hours before infection. In other experiments, HIE monolayers were differentiated 4 days before infection without pretreatment, and infected in the presence of 500 M of glycochenodeoxycholic acid (GCDCA; Sigma) or 500 M GCDCA plus 50 M of a ceramide.
Ten percent fecal filtrates were pre-diluted 1:10, 1:100, 1:1000, and 1:10000 in PBS. Each To determine viral infectivity, we inoculated duplicate plates. After 1 hour of incubation at 37°C and 5% CO2, we washed the monolayers twice with CMGFand added 100 L of differentiation medium containing 1% sow bile, 500 M GCDCA, or 500 M GCDCA plus 50 M ceramide to each well. For each set of infections, 1 plate was immediately frozen at 70°C and a duplicate plate was incubated at 37°C, 5% CO2 for 72 hours and frozen at 70°C. Viral RNA was extracted from input virus, and HIE monolayers at 1 hour postinfection (hpi) and 3 days postinfection (dpi) were determined by RT-qPCR as described below (Technical Appendix Figure 2).

Norovirus Detection, Quantification, and Genotyping
We extracted viral RNA from input virus and HIE monolayers at 1 hpi and 3 dpi using Samples with titer below the limit of detection were arbitrarily assigned to half of the limit of detection (100 RNA copies per 5 L of RNA or 2.0  10 3 RNA copies/well). Positive samples were genotyped by a dual typing RT-PCR using oligonucleotide primer sets specific for GI and GII viruses (5). PCR products were visualized on a 2% agarose gel and purified by ExoSAP-IT (Affymetrix, Cleveland, OH, USA). Genotypes were assigned by phylogenetic analysis using the unweighted-pair group method with reference sequences used by CaliciNet (6) for capsid typing, or the Norovirus Typing Tool, version 2.0 (7,8).

Virus Growth Kinetics
For growth curves, cells versus supernatant, and replace-media experiments, growth kinetics were performed by inoculating HIEs with human norovirus at 3.3-9.3  10 5 copies/well. 1-hour incubation at 37°C, 5% CO2, plates were washed twice with CMGFand 100 L of differentiation medium was added. For each set of infections, 1 plate was immediately frozen at 70°C and the remained plates were incubated at 37°C, 5% CO2 for 72 hours. We determined viral RNA levels in frozen lysates, cells, or supernatants by RT-qPCR. Wells that showed virus replication as an increase in genomic copies per well at 72 hours versus 1 hpi were scored as positive. ID50 was calculated by the Reed-Muench method (9).

Alcohol Treatment
We diluted 10% fecal filtrates 1:10 in 70% ethanol or isopropanol solutions. After incubation for 1 minute or 5 minutes, we neutralized the samples by 1:10 dilution in CMGFsupplemented with 10% FBS. A nontreatment control (10% fecal filtrate diluted 1:100 in PBS) and a neutralization control (10% fecal filtrate diluted 1:10 in PBS and 1:10 in CMGFsupplemented with 10% FBS) were also included. All alcohol solutions were made fresh by addition of the appropriate volume of cell culture grade water (Life Technologies) to molecular biology grade absolute ethanol or isopropanol (Fisher Scientific, Fairlawn, NJ, USA).

Chlorine Suspension Assays
We prepared fresh chlorine stock solutions at 1,000 ppm and 10,000 ppm by dilution of commercial bleach (Clorox, 6% sodium hypochlorite) in cell culture grade water (Life Technologies). For chlorine inactivation experiments, we diluted 20 L of 10% fecal filtrate in an appropriate volume of 10,000 ppm or 1,000 ppm stock solutions to achieve final chlorine concentrations of 5000, 1000, 800, 600, 400, 200, 100, 50, and 5 ppm. After exposure for 1 minute, we added sodium thiosulfate (final concentration: 50 mg/L) to neutralize free chlorine. A nontreatment control (10% fecal filtrate diluted in PBS) and a neutralization control (10% fecal filtrate diluted in PBS and sodium thiosulfate) were also included. All incubations were performed at room temperature. All inactivation experiments and infections were done in triplicate.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). Experiments were performed at least 3 times (3 technical replicates each time) from independent enteroid preparations, as indicated in the figure legends. Data are presented as mean ± SD. Except when specified, a Student's t test was used to determine statistical significance. Specific p values are detailed in the figure legends.