Human noroviruses cause severe, self-limiting gastroenteritis that typically lasts 24–48 hours. Because of the lack of suitable tissue culture or animal models, the true nature of norovirus pathogenesis remains unknown. We show that noroviruses can infect and replicate in a 3-dimensional (3-D), organoid model of human small intestinal epithelium. Cells grown on porous collage-coated beads under fluid shear conditions in rotating wall vessel bioreactors differentiate into 3-D architectures resembling both the morphologic and physiologic function of in vivo tissues. Microscopy, PCR, and fluorescent in situ hybridization provided evidence of norovirus infection. Cytopathic effect and norovirus RNA were detected at each of the 5 cell passages for genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts that used differentiated monolayer cultures failed.

Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.

We evaluated the use of the PCR for detection of enteric viruses in groundwater. To do this, we used an improved sample-processing technique and a large-volume amplification protocol. The objective of this study was to use advanced molecular techniques to develop a rapid and simple method which can be used by the water industry for detection of viral contamination in a variety of water samples. The strategy described here fulfills the water industry’s need for a rapid, reliable, easily performed method for analyzing groundwater for virus contamination. Viruses were detected after concentration from at least 400 gallons (1,512 liters) of water by a filter adsorption and elution method, which resulted in a concentrate containing viruses. A total of 150 samples were analyzed by performing cell culture assays for enteroviruses and by performing reverse transcription PCR (RT-PCR) analyses for enteroviruses, hepatitis A virus, and rotavirus. Thirteen samples (8.7%) produced cellular cytopathic effects when the Buffalo green monkey cell line was used. When primers specific for enteroviruses were used in RT-PCR, 40 of 133 samples (30.1%) tested positive for the presence of enterovirus RNA. When hepatitis A virus-specific primers were used, 12 of 139 samples (8.6%) were considered positive for the presence of hepatitis A viral RNA. The RT-PCR analysis performed with rotavirus-specific primers identified 18 of 130 samples (13.8%) that were positive for rotavirus RNA sequences. Our sample-processing technique and large-volume PCR protocol (reaction volume, 300 μl) resulted in sufficient removal or dilution of inhibitors so that more than 95% of the samples could be assayed by PCR. Because of its sensitivity for detecting viral nucleic acid sequences, PCR analysis should produce more positive results than cell culture analysis. Since either cell culture analysis or PCR can reveal only a “snapshot” of the quality of the groundwater being sampled, PCR seems to be a desirable rapid initial screening tool.