Comparison of Human Intestinal Enteroids and Zebrafish Larvae for GI and GII Human Norovirus Replication

Kristen Gibson, University of Arkansas, Fayetteville

Sahaana Chandran - Department of Food Science, Center for Food Safety, University of Arkansas System Division of Agriculture, Fayetteville, AR, USA Jan Vinjé - Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Kimberly Huynh - Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Kristen E. Gibson - Department of Food Science, Center for Food Safety, University of Arkansas System Division of Agriculture, Fayetteville, AR, USA

Entry and Replication

Human intestinal enteroids (HIE) and zebrafish larvae (ZF) models support the replication of certain strains of human norovirus (HuNoV), the leading cause of viral gastroenteritis worldwide. The replication of 15 HuNoV (8 genotypes) positive stool specimens from patients ranging from 5 months to 83 years old was evaluated in both HIE and ZF models. The yolk of 3-day post-fertilization ZF larvae were microinjected with 3 nL 10% clarified stool suspension. Each day post-infection (dpi), 10 larvae were pooled as one sample, and two samples were collected daily until 5 dpi. Viral RNA was extracted from harvested larvae and quantified using droplet digital PCR. For HIE model, J4 enteroid monolayers were inoculated with 1/100 diluted of each stool specimen. Viral RNA levels were quantified at 1 and 72 hours post-infection using reverse transcription real-time PCR. All genogroup (G) I specimens (n=5) replicated in both models showing a 1 to 2.5 log increase in HuNoV RNA. Of the 10 GII specimens tested, 8 replicated in the ZF model and 7 replicated in the HIE model. Specimens that failed to replicate in each model were not the same, although most of them were GII.4 Sydney [P16]. One additional strain, GII.17[P17], replicated only in the ZF model. Among the GII strains, viral RNA increase ranged from 1.6 to 3.3 log in the ZF and 0.4 to 3.6 log in the HIE model. These data highlight variability in HuNoV replication efficiency across models, potentially indicating the influence of model-specific factors on replication.