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, with a human intestinal enteroid (HIE) culture system where HuNoVs are able to replicate reproducibly, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity. Dasabuvir, 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 A rotavirus (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.
Main text
Human noroviruses (HuNoVs) cause acute gastroenteritis and foodborne diseases among all age groups worldwide. HuNoVs often cause an economic burden to societies due to health care costs and loss of productivity, and therefore pose a public health concern. Noroviruses are non-enveloped viruses possessing a positive sense, single stranded RNA genome whose length is approximately 7.5kb long. They are genetically classified in 10 genogroups (GI-GX) and further divided into 48 genotypes based on their capsid and polymerase gene sequences (Chhabra et al., 2019). Among those, GII.4 genotype is the most frequently distributed and causes outbreak in humans globally (Cannon et al., 2021; Mallory et al., 2019).
Since a robust culture system to allow HuNoV replication was not established for almost 50 years, there is no established treatment options such as vaccines or antiviral regimens available. Recently, several HuNoV successive cultivation models employing a human B cell line (Jones et al., 2014), tissue stem cell-derived human intestinal enteroids (HIEs) (Ettayebi et al., 2016), human induced pluripotent stem cell-derived intestinal organoids (Sato et al., 2019), and zebrafish larvae (Van Dycke et al., 2019) have been developed. The stem cell-derived HIE system is currently used by researchers worldwide to study HuNoV biology and inactivation strategies (Alvarado et al., 2018; Chan et al., 2021; Costantini et al., 2018; Ettayebi et al., 2021; Hosmillo et al., 2020; Lin et al., 2020; Murakami et al., 2020), although, to our knowledge, compound screens for identifying HuNoV antiviral agents have not been reported.
Drug repurposing is a time-saving, affordable strategy to discover new therapeutic uses for approved or developing drugs to treat other disease(s) apart from their original use (Low et al., 2020). This strategy is being widely utilized to establish effective therapeutics for treatment of coronavirus disease 2019 (COVID-19). Indeed, numerous antiviral drugs including remdesivir, ivermectin, or nelfinavir has been identified as promising candidates for SARS-CoV-2 (Low et al., 2020; Watashi, 2021). Here, with the HIE culture system, we screened an antiviral compound library composing 326 bioactive substances, including those targeting influenza virus, human immunodeficiency virus (HIV), or Hepatitis C virus (HCV) to reassess their effect on HuNoV infection.
Three dimensional (3D) HIEs were dissociated, and plated on collagen-coated 96 well plates to prepare two-dimensional (2D) monolayers (Fig. 1A). The cells were then differentiated by culturing them in differentiation medium, which does not include Wnt3A and R-spondin to support HIE’s stemness. The differentiated HIE monolayers were inoculated with GII.4 HuNoV in the presence of each compound dissolved in DMSO for 1 hr at 37°C. DMSO was added to the wells without compound (DMSO control). For this screening step, one well was used to analyze each compound (n = 1). The cells were washed, and cultured in differentiation medium containing the compound for 24 hrs. The infected cells and supernatant were then harvested, and viral replication was evaluated by RT-qPCR analysis to determine the HuNoV RNA genome equivalents (GEs) (Fig. 1B). Cytotoxicity for each compound was also determined by LDH assay. First, to evaluate the reproducibility of our HIE system with respect to HuNoV growth throughout the screening, we plotted the level of viral GEs at 1 or 24 hr post-infection (hpi) from 7 independent experiments that were used for the compound screen. The fold changes of viral GEs between 1 and 24 hpi in each experiment ranged from 65 to 230 (mean ± s.d.; 104.3 ± 58.3). A positive control, 2’-C-Methylcytidine (2-CMC) completely blocked viral infection without any cytotoxicity in all experiments, consistent with a previous report (Fig. 1C and D) (Hosmillo et al., 2020). These results demonstrate that our HIE cultivation system reproducibly supports HuNoV replication, and is suitable for evaluating the effect of the compounds against HuNoV.
We next determined the relative percentages of HuNoV GE and cytotoxicity at 24 hpi by normalizing the data of compound-treated cells to DMSO-treated cells. The screening results were plotted as (%) HuNoV GE vs (%) Cytotoxicity (Fig. 1E). We selected 3 compounds, Dasabuvir (DSB), G243-1637, and 3370-3410, which reduced HuNoV GEs by 95% without cytotoxicity for further validation (Fig. 1E and Table S1). For validation purpose, we repeated the experiment with technical 3 replicates and confirmed the reproducible inhibitory effect of DSB and G243-1637 against HuNoV infection (Fig. S1). We selected DSB which showed strongest inhibitory effect for further studies. DSB has been developed as one of direct-acting anti-HCV drugs which targets HCV NS5B RNA-dependent RNA polymerase (RdRp) (Trivella et al., 2015). So far, there have been no report regarding any antiviral effects on HuNoV.
Next, we performed additional experiments, with DSB at varying concentrations ranging from 3.125 μM to 50 μM to calculate EC50 and CC50 values. DSB treatment alone showed no cytotoxicity, except for the highest concentration (50 μM), which showed a 17% reduction of cellular ATPs (cell viability) or 10% increase of LDH release (cytotoxicity), as compared to the DMSO control (Fig. S2). Again, DSB did not induce cytotoxicity, except at the highest dose (50 μM) in J2 monolayers infected with GII.4 HuNoV, whereas it showed a dose-dependent inhibition of viral replication with an EC50 value of 11.71 μM (Fig. 2A). To ascertain the authenticity of DSB’s inhibitory effect, we repeated the experiment with the identical compound from different resources and found that the results were comparable (Fig. S3). Dose-dependent reduction of viral replication by DSB was also observed in J2 HIE monolayers infected with a different HuNoV strain GII.3 (Fig. 2B). We further assessed DSB’s inhibitory effect using J3 HIEs established from an independent donor following the infection of GII.3 or GII.4 HuNoV and observed the same trends (Fig. 2C and D). Taken together, DSB exerted an inhibitory effect on two HuNoV genotypes and HIEs established from distinct individuals, strongly suggesting that the effect is neither genotype- nor HIE (donor)-dependent.
Next, we tested the effect of DSB on the infection of human A rotavirus (RVA) and SARS-CoV-2, both of which has been previously reported to be able to infect and replicate HIEs (Lamers et al., 2020; Saxena et al., 2016; Zou et al., 2019). Two concentrations of DSB were used in these studies; non-effective (6.25 μM) and effective concentration (20 μM) against HuNoV, respectively. As shown in Fig. 3A and B, DSB showed an antiviral effect (2.75-fold decrease) on RVA infection at a concentration of 20 μM, while it almost completely inhibited (26.9-fold decrease) SARS-CoV-2 infection in J2 HIE monolayers. We also confirmed DSB’s inhibition with no cytotoxicity using VeroE6/TMPRSS2 cells which are highly susceptible to SARS-CoV-2 infection (Figs. 3C, D, and S4) (Matsuyama et al., 2020).
The mechanism of action for the virus inhibitions by DSB remains to be elucidated. DSB is a non-nucleotide inhibitor of HCV NS5B RdRp that likely binds to palm domain of NS5B and thereby prevents elongation of the nascent viral genome (Kati et al., 2015). Therefore, it might also target the RdRp of other viruses such as HuNoV and SARS-CoV-2, possibly because of the presence of conserved sequences being targeted by DSB. Indeed, there is a report showing that DSB partially inhibits RdRp activity of Middle East respiratory syndrome coronavirus (MERS-CoV) (Min et al., 2020). Targeting viral protease might be another scenario for the inhibition; a very recent virtual screening study predicted that dasabuvir has a potential to inhibit 3-chymotrypsin-like protease (3CLPRO) of SARS-CoV-2 (Jade et al., 2021).
With a HCV subgenomic replicon system, DSB inhibits HCV of genotype 1 with EC50 values of < 10 nM (Kati et al., 2015). In contrast, DSB inhibits HuNoV infection with EC50s ranging between 7.55 and 12.41 μM (Fig. 2), which is comparable to its effectiveness to inhibit RdRp activity of MERS-CoV-2 (Min et al., 2020) or infection of vector-borne flaviviruses (Stefanik et al., 2020). This implies that higher concentration is required to exert an antiviral effect on non-HCV viruses, possibly due to lower binding efficiency of DSB to non-HCV RdRp(s) or unknown mechanism of inhibitory action.
In summary, through the screening of an antiviral compound library, we identified DSB as a novel HuNoV inhibitor that warrants further clinical investigation. To our knowledge, this was a first time to identify ‘bona fide’ anti-HuNoV agents using the HIE culture system. Our study also shed light on the usefulness of the HIE platform for investigating of anti-HuNoV agents and/or host factors regulating HuNoV infection, which will contribute to better understanding of HuNoV lifecycle and development of vaccine and antiviral regimens.
Acknowledgements
This study was supported by grants from by the Japan Society for the Promotion of Science KAKENHI Grant JP20K07520 (to T.H.); the Japan Agency for Medical Research and Development (AMED) Grants JP21fk0108149 (to K.M. and T.H.), JP21fk0108102 (to M.M. and K.M.), JP21fk0108121 (to K.M.), JP21wm0225009 (to M.M.), JP21fk0108121 (to Y.F.), and NIH Grant PO1 AI057788 (to M.K.E.). We thank Drs. Hiroyuki Shimizu, Shutoku Matsuyama and Noriyo Nagata (National Institute of Infectious Diseases) for technical assistance.