Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids

Abstract

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2 An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.

Keywords: CRISPR-Cas9; RNA-Seq; enteroids/organoids; human norovirus; interferon.

Fig. 1.

GII.4 HuNoV induces robust IFN-stimulated gene responses in jejunal HIEs. Monolayer (J2 and J11) HIEs were inoculated with media, gamma-irradiated GII.4 (gGII.4) or infectious GII.4 (TCH12-580) stool filtrate containing 1.8 × 10⁸ genome equivalents (GEs) of HuNoV for RNA-Seq analysis. (A) PCA was performed on the resulting normalized log₂ counts per million (cpm) expression matrix after correcting for the HIE genetic background. (B) The number of differentially up- and down-regulated genes (linear fold change of at least 2 and a FDR ≤ 0.05) is summarized in the bar chart. (C and D) Volcano plots and a heatmap of the differential gene analysis illustrate the progression of response with time. Genes were considered differentially expressed if they had a linear fold change of 2 and a FDR ≤ 0.05, and are indicated using a red color in the volcano plot. (E) The up-regulated (top 10) and down-regulated (bottom 10) significant (FDR ≤ 0.25) Reactome pathways are ranked by the normalized enrichment score (NES) based on the changes in gene expression in GII.4-infected compared to gGII.4-inoculated cultures between 6 hpi and 10 hpi, and between 10 hpi and 24 hpi. (F) The statistically significant genes (fold change of at least 2 and a FDR ≤ 0.05 for any time point for the GII.4-infected or gGII.4-inoculated cultures compared to media controls) for selected IFNs and ISGs are visualized as heatmaps. # indicates that the change in IFNA1 was not significantly different.

Fig. 2.

GII.4 HuNoV induces a type III IFN pathway in jejunal HIEs. HIE (J2) monolayers were inoculated with media (mock), GII.4 (TCH12-580) HuNoV (9 × 10⁷ GEs/well), virus mixed with neutralizing antiserum (GII.4 + nAb) or treated with 100 μg/mL of poly(I:C) in the presence of 500 μM glycochenodeoxycholic acid (GCDCA). Poly(I:C) treatment of HIEs for the same time points without GII.4 inoculation served as the positive control. RNA extracted from the cells and medium was used to quantify (A) GII.4 HuNoV GEs, (B) IFNB1 transcripts, (C) IFNL1 transcripts, and (D) ISG IFI44L transcripts. Each data bar represents the mean of three wells of inoculated HIEs, except for the mean of two wells of poly(I:C)-treated HIEs at 24 hpi. Error bars denote SD. * indicates P < 0.05. Each experiment was performed at least two times. For the Luminex assay, J2 and J11 HIEs were inoculated with media, GII.4 (TCH12-580, 9 × 10⁷ GEs/well), GII.3 (TCH04-577, 1.7 × 10⁷ GEs/well), 25 μg/mL of poly(I:C), gamma-irradiated GII.4, or GII.3 HuNoV in the presence of 500 μM GCDCA for 96 hpi. The concentration of IFNB1, IFNL2, and IFNL2/3 proteins (pg/mL) (E) J2 and (F) J11 HIEs are shown. Dashed lines indicate the limit of detection (LOD) for each IFN. Supernatants from two wells for each condition and each HIE line were tested.

Fig. 3.

GII.4 HuNoV replication is sensitive to exogenously activated IFN responses. HIE (J2) monolayers were preincubated with media only (no additive), 1,000 U/mL IFNA1 or IFNB1, or 100 ng/mL IFNL1, IFNL2, or IFNL3 for 24 h, or with 3 μg/mL or 30 μg/mL poly(I:C) at the indicated time points before inoculation with (A–D) 9 × 10⁴ GEs or (E and F) 1.8 × 10⁵ GEs of GII.4 (TCH12-580) stool filtrate (E and F) in the presence of 500 µM GCDCA. Cells were cultured 24 h at 37 °C in the presence of GCDCA without (mock) or with the corresponding IFN (A and C) or with 25 μg/mL of recombinant Y136 (E and F). Transcripts for HuNoV GEs (A, B, and E) or the ISG IFI44L (C, D, and F) were quantified by RT-qPCR. For A and B, each data bar represents the replication efficiency of treated wells relative to untreated wells at 24 hpi. Data were calculated by the mean of three wells of inoculated HIEs. Error bars denote SD. * indicates P < 0.05 and n.s. indicates P ≥ 0.05. Each experiment was performed at least two times.

Fig. 4.

GII.4 HuNoV replication is not increased in IFN pathway-deficient HIEs with attenuated ISG induction. WT, IFNAR1-KO, IFNLR1-KO, STAT1-KO, MAVS-KO, and STAT1/STAT2-DKO HIEs were inoculated with 9 × 10⁴ GEs of GII.4 (TCH12-580) in the presence of 500 µM GCDCA. Cells were cultured for either 24, 48, 72, or 96 h at 37 °C in the presence of GCDCA. RNA was extracted at different time points for RT-qPCR to quantify (A, C, and E) GII.4 HuNoV GEs or (B, D, and F) ISG IFI44L transcripts. Each data bar represents the mean of three wells of inoculated HIEs. Error bars denote SD. * indicates P < 0.05. Each experiment was performed at least two times.

Fig. 5.

Exogenous IFN reduces GII.3 HuNoV replication in HIEs. HIE (J2) monolayers were preincubated with media only (no additive), 1,000 U/mL IFNA1 or IFNB1, or 100 ng/mL IFNL1 or IFNL3 for 24 h, then inoculated with (A and B) 4.3 × 10⁵ GEs or (C and D) 8.6 × 10⁵ GEs of GII.3 (TCH04-577) stool filtrate in the presence of 500 μM GCDCA. Cells were cultured 24 h at 37 °C in the presence of GCDCA without or with the corresponding IFN. RNA was extracted at different time points for RT-qPCR to quantify (A and C) GII.3 HuNoV GEs or (B and D) ISG IFI44L transcripts. Data were calculated by the mean of three wells of inoculated HIEs. Error bars denote SD. * indicates P < 0.05 and n.s. indicates P ≥ 0.05. Each experiment was performed at least two times.

Fig. 6.

GII.3 HuNoV replication is increased in IFN pathway-deficient HIEs. WT, IFNAR1-KO, STAT1-KO, MAVS-KO, and STAT1/2-DKO HIEs were inoculated with 4.3 × 10⁵ GEs of GII.3 (TCH04-577) in the presence of 500 µM GCDCA. Cells were cultured for either 24, 48, 72, or 96 h at 37 °C in the presence of 500 μM GCDCA. RNA was extracted at different time points for RT-qPCR to quantify (A, C, and E) GII.3 HuNoV GEs for the replication kinetics or (B, D, and F) ISG IFI44L transcripts. Each data bar represents the mean of three wells of inoculated HIEs. Error bars denote SD. * indicates P < 0.05. Each experiment was performed at least three times.

Fig. 7.

STAT1-KO HIE cells are more susceptible to GII.3 infection. HIE monolayers were inoculated with 4.3 × 10⁶ GEs of GII.3 (TCH04-577) (A–C), or the indicated GEs/well (D) in the presence 500 µM GCDCA. Cells were cultured in the presence of GCDCA for indicated times for FFA (A–C) or (D) 7 d for TCID₅₀. (A) GII.3-positive cells (green) were detected after staining with guinea pig anti-HuNoV Ab with nuclei (blue) stained by DAPI. Infected cells in clusters identified viral spreading. (B) Numbers of infected cells were calculated in six independent images per condition and each experiment was performed in triplicate (n = 18 in total). (C) The frequency of foci of different sizes was determined from six independent images per condition at 48 and 72 hpi in triplicate experiments (n = 18 in total). (D) Fold changes of GII.3 RNA replication at 7 d compared to 1 hpi and the numbers of positive wells at each inoculum dose were used to determine the TCID₅₀ in WT and STAT1-KO cultures. * indicates P < 0.05 and n.s. indicates P ≥ 0.05. Each experiment was performed at least two times.