Dasabuvir Inhibits Human Norovirus Infection in Human Intestinal Enteroids

Abstract

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity using a human intestinal enteroid (HIE) culture system in which HuNoVs are able to replicate reproducibly. Dasabuvir (DSB), which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human rotavirus A (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs, and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation. IMPORTANCE Although there is an urgent need to develop effective antiviral therapy directed against HuNoV infection, compound screening to identify anti-HuNoV drug candidates has not been reported so far. Using a human HIE culture system, our compound screening successfully identified dasabuvir as a novel anti-HuNoV inhibitor. Dasabuvir's inhibitory effect was also demonstrated in the cases of SARS-CoV-2 and RVA infection, highlighting the usefulness of the HIE platform for screening antiviral agents against various viruses that target the intestines.

Keywords: acute gastroenteritis; antiviral drug; compound screen; dasabuvir; intestinal enteroids; norovirus.

FIG 1

Screening for antiviral compounds that inhibit human norovirus (HuNoV) infection in human intestinal enteroids (HIEs). (A and B) Schematic illustrations of compound screening. Three-dimensional HIEs (J2) were dissociated into single cells using TrypLE enzyme and plated in a 96-well plate to culture them as 2D monolayers. Differentiated HIE monolayers were then inoculated with GII.4 HuNoV-containing stool filtrates in the presence of the compounds (10 μM, n = 1). After 1 h incubation at 37°C, the cells were washed and then cultured in differentiation medium containing the compounds (10 μM) in a 100-μl volume until 24 h postinfection (hpi). Viral RNA extracted from the cells and 75 μl of supernatant at 1 or 24 hpi were subjected to reverse transcription-quantitative PCR (RT-qPCR) to measure viral genome equivalents (GEs). The rest of the supernatant was subjected to lactate dehydrogenase (LDH) assay to measure cytotoxicity. (C) HuNoV replication in J2 monolayers throughout the screening. We performed 7 experiments to screen all 326 compounds. Dimethyl sulfoxide (DMSO) and 2′-C-methylcytidine (2-CMC; 389 μM) were used as the controls in every test. Viral GEs in DMSO and 2-CMC-treated samples at 24 hpi were normalized to the DMSO control at 1 hpi. **, P < 0.01 versus DMSO control at 24 hpi, one-way analysis of variance (ANOVA) followed by Dunnett's multiple-comparison test. (D) Cytotoxicity of 2-CMC in J2 monolayers at 24 hpi. Results were normalized to the DMSO control. (E) Scatterplot of the % HuNoV GEs versus % cytotoxicity for all tested compounds. Results were normalized to the DMSO control. DSB, dasabuvir.

FIG 2

Effect of dasabuvir on HuNoV infection in HIE monolayers. J2 (A and B) or J3 (C and D) HIE monolayers were inoculated with GII.4 (A and C) or GII.3 (B and D) HuNoV-containing stool filtrate in the presence of DSB at the indicated concentrations and were cultured until 20 hpi. The percentages of HuNoV GEs (red lines) and cytotoxicity (blue lines) were determined as in Fig. 1 and were normalized to the DMSO control. Values represent the mean ± standard deviation (SD) (n ≥ 6). EC₅₀, 50% effective concentration; CC₅₀, 50% cytotoxic concentration.

FIG 3

Dasabuvir inhibits SARS-CoV-2 infection in J2 HIE monolayers and VeroE6/TMPRSS2 cells. (A and B) J2 HIE monolayers were inoculated with RVA (A) and SARS-CoV-2 (B) in the presence of the indicated compounds and were cultured until 20 hpi. 2-CMC (389 μM) or remdesivir (RDV; 10 μM) were used as positive controls. The percentages of viral GEs were determined by RT-qPCR, and were normalized to the DMSO control at 20 hpi. Values represent the mean ± SD (n ≥ 5). (C and D) VeroE6/TMPRSS2 cells were left uninfected or were infected with SARS-CoV-2 for 20 h in the presence of the indicated compounds. The cells were then stained with anti-SARS-CoV-2 spike (S) RBD monoclonal antibody and 4′,6-diamidino-2-phenylindole (DAPI), followed by imaging analysis. (C) Representative fluorescence images showing SARS-CoV-2 S protein (green) and cell nucleus (blue). Bar, 200 μm. (D) The percentages of infected cells were normalized to those of DMSO-treated cells infected with SARS-CoV-2 at 20 hpi. Values represent the mean ± SD (n ≥ 8). **, P < 0.01 versus DMSO control at 20 hpi, one-way ANOVA followed by Dunnett’s multiple-comparison test. n.s., not significant (P > 0.05).