Novel Use of Porcine Gastric Mucin-Coated Beads to Enrich Infectious Human Norovirus Prior to Infection of Human Intestinal Enteroids

Samantha Wales, FDA

Samantha Q. Wales, FDA Human Foods Program, Office of Applied Microbiology and Technology, Virology and Parasitology Branch Cameron Boerner, FDA Human Foods Program, Office of Applied Microbiology and Technology, Virology and Parasitology Branch

Entry and Replication

While norovirus research was significantly boosted with the recent discovery of human intestinal enteroids (HIEs)’ ability to support viral replication, the system still suffers from severe limitations including cost, complexity, difficulty obtaining samples, preference for certain strains, and further complicated by the requirement of high titer virus for successful infection. Our laboratory has previously demonstrated that we can successfully recover infectious norovirus from food samples, but the amount of virus necessary to spike food samples for effective recovery does not reflect the small amounts typically present in food samples that cause illness. Thus, there is a need to amplify the recovery of infectious virus particles from food matrices. Porcine gastric mucin (PGM)-coated beads have been used in the past as a method to isolate virus particles with intact capsids from food and environmental samples, coupled with RT-qPCR. We wanted to determine if this approach could be applied to HIEs. Equal volumes of stool suspensions of sixteen norovirus strains (12 GII and 4 GI) were diluted in media or PBS-0.05% Tween for -beads or +beads, respectively. Samples -beads remained on ice and +beads were mixed with PGM-coated beads and incubated with rocking. After bead capture and wash steps to remove unbound virus, infections were performed in parallel. The results confirm that HIEs can support infection with norovirus bound to PGM-coated beads without toxicity and indicate that PGM-coated beads enrich for intact virions, as evidenced by increased binding at 0h in the +beads samples as compared to -beads.