Human Intestinal Enteroids Used to Detect Norovirus Infectivity

Molecular nucleic acid tests, such as real-time reverse transcription polymerase chain reaction (rRT-PCR), are widely used for laboratory diagnosis of norovirus RNA in clinical samples.

These molecular assays are virus specific and their analytical sensitivity is high, but they cannot distinguish between infectious and noninfectious viruses. A recently developed human intestinal enteroid (HIE) culture system (cultures that contain multiple intestinal epithelial cell types) for human norovirus correlates between viral load and virus infectivity.

Scientists at the Chinese University of Hong Kong (Hong Kong, China) examined the infectivity of human pandemic norovirus genogroup II genotype 4 (GII.Pe-GII.4 Sydney) strains at different inoculating levels by using the adult stem cell–derived HIE line J2. The team used fecal samples from three norovirus-positive children and from adults in the norovirus surveillance program in Hong Kong.

The investigators measured norovirus RNA levels in supernatant at 1, 24, and 72 hours after inoculation by using rRT-PCR with a 10-fold serially diluted standard of in vitro–transcribed norovirus RNA. They considered a >10-fold increase in RNA level at 72 hours after inoculation from baseline (1 hour after inoculation) to indicate productive viral replication and to confirm the presence of infectious virus. They subjected fecal filtrate dilutions to norovirus antigen detection by use of the commercial RIDASCREEN Norovirus 3rd Generation EIA.

The team reported that from 2014 through 2018, a total of 114 (6.5%) of 1,754 norovirus-positive fecal samples from patients admitted to the Prince of Wales Hospital, with acute gastroenteritis had Ct values >30 (Ct median 17.8; interquartile range 14.8–22.3; range 5.5–39.2). Among the 1,579 (90.0%) genotyped samples, the proportion of GII.4 was 49.1%; other GII, 44.8%; GI, 5.2%; and co-infections with >1 norovirus capsid genotype, 0.9%. Analytical sensitivity of virus replication in HIEs for measuring moderate norovirus shedding was higher than that of EIA by being able to detect infectious virus in fecal filtrate dilutions with Ct values of 25–30.

The authors concluded that they had demonstrated that a Ct cutoff of 30 for a widely used clinical diagnostic rRT-PCR can indicate the presence of infectious GII.Pe-GII.4 Sydney norovirus in an HIE culture model. Patients shedding low levels of norovirus RNA may not be infectious, which should be considered both for estimation of attributable norovirus burden and for clinical management of viral gastroenteritis. The study was published in the September 2019 issue of the journal Emerging Infectious Diseases.

Related Links:
Chinese University of Hong Kong