Insights into human norovirus cultivation in human intestinal enteroids

Abstract

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights into this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells that were then transplanted and matured in mice before making enteroids (H9tHIEs), genetically engineered (J4FUT2 knock-in [KI], J2STAT1 knockout [KO]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of genogroup I and II HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research.IMPORTANCEHuman noroviruses (HuNoVs) cause global diarrheal illness and chronic infections in immunocompromised patients. This paper reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from (i) different intestinal segments of single donors, (ii) human embryonic stem cell-derived organoids transplanted into mice, (iii) genetically modified lines, and (iv) a patient with common variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment, and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically modified J4FUT2 knock-in (KI) HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

Keywords: HuNoV cultivation; enteroids; organoid growth media; organoids.

Fig 1

HuNoV replication is enhanced in HIEs plated in OGM compared to ODM. 3D J4FUT2-KI HIEs were propagated in L-WRN media. Monolayers were seeded in OGM proliferation medium with ROCK inhibitor and were differentiated in OGMd or ODM. Differentiated monolayers were inoculated with GII.4 Sydney[P31] (9 × 10⁵ GEs/well) or GII.3[P21] (4.3 × 10⁵ GEs/well) diluted in CMGF(–) with 500 µM GCDCA. After 1 hpi, monolayers were washed twice and cultured in the OGMd or ODM (+500 µM GCDCA). Values above the bars represent net log₁₀ difference in viral growth (Δ24h-1h). Gray shading indicates GE at 1 hpi and purple shading shows GE at 24 hpi. Mean data compiled from two independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol shapes (circle or triangle). Significance was determined at 24 hpi using Student’s t-test. ***P value < 0.001.

Fig 2

HuNoV replication in H9tHIEs. Differentiated H9tHIE monolayers in OGM differentiation medium were infected with (A) GII.4 Sydney[P31] (9 × 10⁵ GEs/well) or (B) GII.3[P21] (4.3 × 10⁵ GEs/well) in the presence of 500 µM GCDCA. After 1 hpi, monolayers were washed twice and cultured in OGM differentiation medium (+500 µM GCDCA). Values on bars represent the net log₁₀ difference in viral growth (Δ24h-1h). Gray shading indicates GE at 1 hpi, and blue (A) or green (B) shows GE at 24 hpi. Mean data compiled from two independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted by different symbol shapes (circle or triangle). Significance was determined at 24 hpi. ***P value < 0.001; **P < 0.01; *P < 0.05.

Fig 3

HuNoV replication in D₂₀₁ HIEs. D₂₀₁ HIE monolayers were differentiated in OGM differentiation medium and infected with GII.4/Sydney (9 × 10⁵ GEs/well), GII.3 (4.3 × 10⁵ GEs/well), GII.6/TCH13-106 (3.3 × 10⁵ GEs/well), or GII.6/CVID (BCM18-1) (9.7 × 10⁴ GEs/well) diluted in CMGF(–) supplemented with 500 µM GCDCA. After 2 hpi, monolayers were washed twice and cultured in OGM differentiation medium (+500 µM GCDCA). Values on bars represent net log₁₀ difference in viral growth (Δ24h-1h). Gray shading indicates GE at 2 hpi, and blue shows GE at 24 hpi. Mean data compiled from two independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted by different symbol shapes (circle or triangle). Significance was determined at 24 hpi. ***P value < 0.001.

Fig 4

HuNoV replication in genetically modified HIEs. HIE monolayers were differentiated in OGM differentiation medium and inoculated with (A) GII.6/CVID (9.7 × 10⁴ GEs/well) or (B) GII.6/TCH13-106 (3.3 × 10⁵ GEs/well) in the presence of 500 µM GCDCA. After 1–2 hpi, monolayers were washed twice and cultured in OGM differentiation medium (+500 µM GCDCA). Values on bars represent net log₁₀ difference in viral growth (Δ24h-1h). Gray bars indicate GE at 2 hpi, and colored bars show GE at 72 hpi. Mean data compiled from two independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted by different symbol shapes (circle or triangle). Significance was determined at 24 hpi. ***P value < 0.001; **P < 0.01.

Fig 5

HuNoV replication in HIEs from adults and infants. HIE monolayers were differentiated in OGM differentiation medium and infected with (A) GII.4/Sydney (9 × 10⁵ GEs/well) or (B) GII.3 (4.3 × 10⁵ GEs/well) diluted in CMGF(–) supplemented with 500 µM GCDCA. After 1 hpi, monolayers were washed twice and cultured in OGM differentiation medium (+500 µM GCDCA). Values on bars represent net log₁₀ difference in viral growth (Δ24h-1h). Gray bars indicate GE at 1 hpi, and colored bars show GE at 24 hpi. Mean data compiled from two independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted by different symbol shapes (circle or triangle). Significance was determined at 24 hpi with significant differences between groups shown. ***P value < 0.001; **P < 0.01.