A Standardized Antiviral Pipeline for Human Norovirus in Human Intestinal Enteroids Demonstrates No Antiviral Activity of Nitazoxanide

Abstract

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis. In immunocompetent hosts, symptoms usually resolve within three days; however, in immunocompromised persons, HuNoV infection can become persistent, debilitating, and sometimes life-threatening. There are no licensed therapeutics for HuNoV due to a near half-century delay in its cultivation. Treatment for chronic HuNoV infection in immunosuppressed patients anecdotally includes nitazoxanide, a broad-spectrum antimicrobial licensed for treatment of parasite-induced gastroenteritis. Despite its off-label use for chronic HuNoV infection, nitazoxanide has not been clearly demonstrated to be an effective treatment. In this study, we established a standardized pipeline for antiviral testing using multiple human small intestinal enteroid (HIE) lines representing different intestinal segments and evaluated whether nitazoxanide inhibits replication of 5 HuNoV strains in vitro . Nitazoxanide did not exhibit high selective antiviral activity against any HuNoV strains tested, indicating it is not an effective antiviral for norovirus infection. HIEs are further demonstrated as a model to serve as a pre-clinical platform to test antivirals against human noroviruses to treat gastrointestinal disease.

Figure 1:

Parameters measured to optimize assessment of antiviral treatment of HuNoV in HIEs. A) Schematic representation of conditions used for standardizing HuNoV antiviral testing in HIEs. After determining the TCID₅₀ for each genotype in each HIE line, 100 TCID₅₀ of each virus was used to assess antiviral activity with different concentrations of compounds. Cytotoxicity was evaluated in tandem with each antiviral assay. B) Bright field and fluorescent microscopy images of vehicle (1% DMSO) and 32 and 100 μM NTZ treated J2 HIEs after 24 h. Cell nuclei were stained with DAPI (blue) and dead cells were stained with propidium iodide (PI) (red). C) Effect of NTZ on HuNoV adsorption to HIE monolayers. 100 TCID₅₀ of 4 HuNoV genotypes were added to either J2 (GII.4) or J4FUT2 (GI.1, GII.3, GII.17) HIEs for 1 or 2 h and genome equivalents were quantitated. Images were taken at 40X magnification. Scale bar = 200 µM. ns = not significant. Data are compiled from n = 2 experiments.

Figure 2:

HIEs are useful models for antiviral studies with HuNoV. J2 HIEs were treated with 5 ascending doses of 2’CMC and the vehicle (2% H₂O) for 24 hours. A) Replication after 24 hours of GII.4 Sydney with 2’CMC or vehicle treatment B) Percent inhibition by 24 hours of GII.4 Sydney by 2’CMC. C) Percent viability was assessed in the J2 HIE line after 24 h. Based on EC₅₀ and CC₅₀ (panels B and C, respectively), the SI was calculated as 31.6. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Data are compiled from n = 2 experiments.

Figure 3.

NTZ inhibition of GII.4-Sydney HuNoV replication in jejunal HIEs parallels its cytotoxicity. J2 HIEs were treated with 5 ascending doses of NTZ and the vehicle (1% DMSO) for 24 hours. A) Replication of GII.4 Sydney with NTZ treatment. B) Percent inhibition of GII.4 Sydney by NTZ. C) Percent viability was assessed in the J2 HIE line after 24 h. Based on EC₅₀ and CC₅₀ (panels B and C, respectively), the SI was calculated as 1.9. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Data are compiled from n = 3 experiments.

Figure 4.

NTZ minimally inhibits GII.3 HuNoV in jejunal HIEs. J4FUT2 HIEs were treated with 5 ascending doses of NTZ and the vehicle (1% DMSO) for 24 hours. A) Replication of GII.3 with NTZ treatment B) Percent inhibition of GII.3 by NTZ. C) Percent viability was assessed in the J4FUT2 HIE line after 24 h. Based on EC₅₀ and CC₅₀ (panels B and C, respectively), the SI was calculated as 4.5. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Data are compiled from n = 4 experiments.

Figure 5.

NTZ inhibition of GII.17 HuNoV replication in jejunal HIEs parallels its cytotoxicity. J4FUT2 HIOs were treated with 5 ascending doses of NTZ and the vehicle (1% DMSO) for 24 hours. A) Replication of GII.17 with NTZ treatment B) Percent inhibition of GII.17 by NTZ. C) Percent viability was assessed in the J4FUT2 HIE line after 24 h. Based on EC₅₀ and CC₅₀ (panels B and C, respectively), the SI was calculated as 1.9. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Data are compiled from n = 3 experiments.

Figure 6.

NTZ inhibition of GI.1 HuNoV replication in jejunal HIEs parallels its cytotoxicity. J4FUT2 HIEs were treated with 5 ascending doses of NTZ and the vehicle (1% DMSO) for 24 hours. A) Replication of GI.1 with NTZ treatment B) Percent inhibition of GI.1 by NTZ. C) Percent viability was assessed in the J4FUT2 HIE line after 24 h. Based on EC₅₀ and CC₅₀ (panels B and C, respectively), the SI was calculated as 2.7. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Data are compiled from n = 2 experiments.

Figure 7.

NTZ inhibition of GII.4 HuNoV replication in small intestinal HIEs parallels its cytotoxicity. J2, D2004, J2004, and IL2004 HIEs were treated with 5 ascending doses of NTZ and the vehicle (1% DMSO) for 24 hours. A) Replication of GII.4 Sydney with NTZ treatment in each HIE line. B) Percent inhibition of GII.4 Sydney by NTZ in each HIE line. C) Percent viability was assessed in the J2, D2004, J2004, and IL2004 HIE lines after 24 h. Based on EC₅₀ and CC₅₀ (panels B and C, respectively), the follow SI values were calculated: J2 = 4.0; D2004 = 1.2; J2004 = 1.2 and IL2004 =1.4. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Data are compiled from n = 2 experiments.